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Sample records for acids lysine methionine

  1. Correlation of carnitine levels to methionine and lysine intake.

    PubMed

    Krajcovicová-Kudlácková, M; Simoncic, R; Béderová, A; Babinská, K; Béder, I

    2000-01-01

    Plasma carnitine levels were measured in two alternative nutrition groups--strict vegetarians (vegans) and lactoovovegetarians (vegetarians consuming limited amounts of animal products such as milk products and eggs). The results were compared to an average sample of probands on mixed nutrition (omnivores). Carnitine levels were correlated with the intake of essential amino acids, methionine and lysine (as substrates of its endogenous synthesis), since the intake of carnitine in food is negligible in the alternative nutrition groups (the highest carnitine content is in meat, lower is in milk products, while fruit, cereals and vegetables contain low or no carnitine at all). An average carnitine level in vegans was significantly reduced with hypocarnitinemia present in 52.9% of probands. Similarly, the intake of methionine and lysine was significantly lower in this group due to the exclusive consumption of plant proteins with reduced content of these amino acids. Carnitine level in lactoovovegetarians was also significantly reduced, but the incidence of values below 30 micromol/l was lower than in vegans representing 17.8% vs. 3.3% in omnivores. Intake of methionine and lysine was also significantly reduced in this group, but still higher compared to vegans (73% of protein intake covered by plant proteins). Significant positive correlation of carnitine levels with methionine and lysine intake in alternative nutrition groups indicates that a significant portion of carnitine requirement is covered by endogenous synthesis. Approximately two thirds of carnitine requirement in omnivores comes from exogenous sources. The results demonstrate the risks of alternative nutrition with respect to the intake of essential amino acids, methionine and lysine, and with respect to the intake and biosynthesis of carnitine. PMID:11043928

  2. Correlation of carnitine levels to methionine and lysine intake.

    PubMed

    Krajcovicová-Kudlácková, M; Simoncic, R; Béderová, A; Babinská, K; Béder, I

    2000-01-01

    Plasma carnitine levels were measured in two alternative nutrition groups--strict vegetarians (vegans) and lactoovovegetarians (vegetarians consuming limited amounts of animal products such as milk products and eggs). The results were compared to an average sample of probands on mixed nutrition (omnivores). Carnitine levels were correlated with the intake of essential amino acids, methionine and lysine (as substrates of its endogenous synthesis), since the intake of carnitine in food is negligible in the alternative nutrition groups (the highest carnitine content is in meat, lower is in milk products, while fruit, cereals and vegetables contain low or no carnitine at all). An average carnitine level in vegans was significantly reduced with hypocarnitinemia present in 52.9% of probands. Similarly, the intake of methionine and lysine was significantly lower in this group due to the exclusive consumption of plant proteins with reduced content of these amino acids. Carnitine level in lactoovovegetarians was also significantly reduced, but the incidence of values below 30 micromol/l was lower than in vegans representing 17.8% vs. 3.3% in omnivores. Intake of methionine and lysine was also significantly reduced in this group, but still higher compared to vegans (73% of protein intake covered by plant proteins). Significant positive correlation of carnitine levels with methionine and lysine intake in alternative nutrition groups indicates that a significant portion of carnitine requirement is covered by endogenous synthesis. Approximately two thirds of carnitine requirement in omnivores comes from exogenous sources. The results demonstrate the risks of alternative nutrition with respect to the intake of essential amino acids, methionine and lysine, and with respect to the intake and biosynthesis of carnitine.

  3. Fortifying plants with the essential amino acids lysine and methionine to improve nutritional quality.

    PubMed

    Galili, Gad; Amir, Rachel

    2013-02-01

    Humans, as well as farm animals, cannot synthesize a number of essential amino acids, which are critical for their survival. Hence, these organisms must obtain these essential amino acids from their diets. Cereal and legume crops, which represent the major food and feed sources for humans and livestock worldwide, possess limiting levels of some of these essential amino acids, particularly Lys and Met. Extensive efforts were made to fortify crop plants with these essential amino acids using traditional breeding and mutagenesis. However, aside from some results obtained with maize, none of these approaches was successful. Therefore, additional efforts using genetic engineering approaches concentrated on increasing the synthesis and reducing the catabolism of these essential amino acids and also on the expression of recombinant proteins enriched in them. In the present review, we discuss the basic biological aspects associated with the synthesis and accumulation of these amino acids in plants and also describe recent developments associated with the fortification of crop plants with essential amino acids by genetic engineering approaches.

  4. Pig performance increases with the addition of DL-methionine and L-lysine to ensiled cassava leaf protein diets.

    PubMed

    Ly, Nguyen Thi Hoa; Ngoan, Le Duc; Verstegen, Martin Wilhelmus Antonius; Hendriks, Wouter Hendrikus

    2012-01-01

    Two studies were conducted to determine the impact of supplementation of diets containing ensiled cassava leaves as the main protein source with synthetic amino acids, DL-methionine alone or with L-lysine. In study 1, a total of 40 pigs in five units, all cross-breds between Large White and Mong Cai, with an average initial body weight of 20.5 kg were randomly assigned to four treatments consisting of a basal diet containing 45% of dry matter (DM) from ensiled cassava leaves (ECL) and ensiled cassava root supplemented with 0%, 0.05%, 0.1% and 0.15% DL-methionine (as DM). Results showed a significantly improved performance and protein gain by extra methionine. This reduced the feed cost by 2.6%, 7.2% and 7.5%, respectively. In study 2, there were three units and in each unit eight cross-bred (Large White × Mong Cai) pigs with an initial body weight of 20.1 kg were randomly assigned to the four treatments. The four diets were as follows: a basal diet containing 15% ECL (as DM) supplemented with different amounts of amino acids L-lysine and DL-methionine to the control diet. The results showed that diets with 15% of DM as ECL with supplementation of 0.2% lysine +0.1% DL-methionine and 0.1% lysine +0.05% DL-methionine at the 20-50 kg and above 50 kg, respectively, resulted in the best performance, protein gain and lowest costs for cross-bred (Large White × Mong Cai) pigs. Ensiled cassava leaves can be used as a protein supplement for feeding pigs provided the diets contain additional amounts of synthetic lysine and methionine.

  5. Short communication: Supplementing lysine and methionine in a lactation diet containing a high concentration of wet corn gluten feed did not alter milk protein yield.

    PubMed

    Mullins, C R; Weber, D; Block, E; Smith, J F; Brouk, M J; Bradford, B J

    2013-08-01

    Primiparous (n=33) and multiparous (n=63) lactating Holstein cows (186±51 d in milk) were used to evaluate the effects of supplementing metabolizable amino acids using lysine in a matrix of Ca salts of fatty acids (Megamine-L, Arm & Hammer Animal Nutrition, Princeton, NJ) and the isopropyl ester of 2-hydroxy-4-(methylthio) butanoic acid (MetaSmart, Adisseo Inc., Antony, France) in diets containing >26% wet corn gluten feed (dry matter basis). Cows were blocked by production level, parity, and pregnancy status, then randomly assigned to 1 of 8 pens and allowed a 7-d adaption period before receiving dietary treatments for 28 d. Pens were assigned randomly to either of 2 diets formulated to differ by metabolizable amino acid supply. Dry matter intake and production were monitored daily and milk components analyzed 3d/wk. Data were analyzed using mixed models with repeated measures. The original design of the study consisted of a control diet predicted to be deficient in lysine and methionine; however, after ingredient nutrients were analyzed and modeled with animal requirements at dry matter intake [26.6±0.35 kg/d (mean ± SEM)] and milk production levels achieved during the study (40.1±0.46 kg/d), only marginal deficiencies were predicted for the control (-8.1g/d for lysine; -1g/d for methionine) according to the National Research Council method, whereas the Cornell Net Carbohydrate and Protein System 5.0 and 6.1 models indicated positive balances for these amino acids (25.9 and 21.8 g/d for lysine, 14.7 and 18.9 g/d for methionine, respectively). Supplementing 30 g/d of metabolizable lysine in a Ca soap matrix and 2.4 g/d of metabolizable methionine as 2-hydroxy-4-(methylthio) butanoic acid led to positive predicted lysine and methionine balances by all 3 models, and predicted metabolizable lysine-to-methionine ratios ranging from 2.9 to 3.1. No treatment effects were observed for dry matter intake, milk yield, milk component concentrations or yields, or energy

  6. Rumen-protected methionine and lysine: effects on milk production and plasma amino acids of dairy cows with reference to metabolisable protein status.

    PubMed

    Awawdeh, Mofleh S

    2016-05-01

    Two experiments were conducted to study the effects of rumen-protected Met (RPM) alone or with rumen-protected Lys (RPL) on milk yield and plasma amino acids of dairy cows. In experiment 1, 24 multiparous Holstein cows (154 DIM) were assigned to one of 3 groups where each cow received 0 g/d of RPM and RPL (C), 30 g/d of RPM (M), or 30 g/d of RPM plus 25 g of RPL (ML). The study lasted for 8 weeks where milk yield and composition were determined weekly. Daily milk yield averaged 28·0, 27·8, and 29·7 kg/cow for the C, M, and ML groups, respectively. Dietary treatments had no effects (P ≥ 0·54) on milk contents of fat, lactose, solid non-fat or total solids. Milk protein content in the ML group was greater (P < 0·05) than the C and M groups. Plasma levels of all AA were not significantly (P ≥ 0·09) affected by supplemental RPL and/or RPM. In experiment 2, 30 multiparous Holstein cows (100 DIM) were assigned to one of 3 groups where each cow received 0 g/d of RPM and RPL (C), 50 g/d of RPM (M), or 50 g/d of RPM plus 25 g/d of RPL (ML). The study lasted for 5 weeks. Cows in the M (30·5 kg) and ML (31·4 kg) groups produced (P < 0·05) more milk than those of the C group (29·1 kg). Under conditions of this study, RPM plus RPL improved milk yield and protein contents of dairy cows and was better than supplying RPM alone. Response in milk yield to RPM and RPL was affected by the MP status of cows which deserves further investigation.

  7. Histone H3 lysine-to-methionine mutants as a paradigm to study chromatin signaling

    PubMed Central

    Herz, Hans-Martin; Morgan, Marc; Gao, Xin; Jackson, Jessica; Rickels, Ryan; Swanson, Selene K.; Florens, Laurence; Washburn, Michael P.; Eissenberg, Joel C.; Shilatifard, Ali

    2015-01-01

    Histone H3 lysine27-to-methionine (H3K27M) gain-of-function mutations occur in highly aggressive pediatric gliomas. Here, we establish a Drosophila animal model for the pathogenic histone H3K27M mutation and show that its overexpression resembles Polycomb repressive complex 2 (PRC2) loss-of-function phenotypes, causing de-repression of PRC2 target genes and developmental perturbations. Similarly, a H3K9M mutant depletes H3K9 methylation levels and suppresses position-effect variegation in various Drosophila tissues. The histone H3K9 demethylase KDM3B/JHDM2 associates with H3K9M nucleosomes and its overexpression in Drosophila results in loss of H3K9 methylation levels and heterochromatic silencing defects. Here we establish histone lysine-to-methionine mutants as robust in vivo tools for inhibiting methylation pathways that also function as biochemical reagents for capturing site-specific histone-modifying enzymes, thus providing molecular insight into chromatin-signaling pathways. PMID:25170156

  8. Estimation of the true ileal digestible lysine and sulfur amino acid requirement and comparison of the bioefficacy of 2-hydroxy-4-(methylthio)butanoic acid and DL-methionine in eleven- to twenty-six-kilogram nursery pigs.

    PubMed

    Yi, G F; Gaines, A M; Ratliff, B W; Srichana, P; Allee, G L; Perryman, K R; Knight, C D

    2006-07-01

    Three experiments were conducted to determine the true ileal digestible (TID) Lys and sulfur AA (SAA) requirement and to compare the bioefficacy of 2-hydroxy-4-(methylthio)butanoic acid (HMTBA) and dl-MET as Met sources in nursery pigs. Experiment 1 included 2 studies: 1 was 662 nursery pigs (Triumph 4 x PIC C22; initial BW 12.2 +/- 0.18 kg) allotted to 1 of 5 dietary treatments with TID Lys concentrations ranging from 1.10 to 1.50%; and the second study was 665 nursery pigs (Triumph 4 x PIC C22; initial BW 12.3 +/- 0.18 kg) allotted to 1 of 5 dietary treatments with TID SAA concentration ranging from 0.63 to 0.90%. In Exp. 2, 638 nursery pigs (Triumph 4 x PIC C22; initial BW 13.0 +/- 0.16 kg) were allotted to the same 5 SAA dietary treatments as in Exp. 1. In Exp. 3, 1,232 pigs (Triumph 4 x PIC C22; initial BW 11.0 +/- 0.30 kg) were allotted to 1 of 7 dietary treatments. The basal diet (diet 1) was supplemented with high concentrations of synthetic AA but no Met; this resulted in a dietary concentration of TID Lys of 1.30% and TID SAA of 0.50%. Diets 2 to 7 were the basal diet supplemented with 3 equimolar levels of HMTBA or dl-MET to provide TID SAA concentrations of 0.56, 0.62, and 0.68%, respectively. In Exp. 1, increasing TID Lys from 1.10 to 1.50% increased ADG (quadratic; P < 0.05) and improved G:F (linear; P < 0.002). The pooled data of Exp. 1 (SAA study) and Exp. 2 indicated that increasing TID SAA from 0.63 to 0.90% increased ADG (quadratic; P < 0.01) and improved G:F (quadratic; P < 0.01). Various methods of analyzing the growth response surface indicated that the optimal TID Lys concentration ranged from 1.28 to 1.32% for ADG (Exp. 1), and the optimal TID SAA concentration ranged from 0.73 to 0.77% for ADG and 0.80 to 0.83% for G:F (pooled Exp. 1 and 2), respectively. In Exp. 3, increasing TID SAA concentrations from 0.50 to 0.68% resulted in a linear improvement of ADG (P < 0.001), ADFI (P < 0.05), and G:F (P < 0.001). The best fit comparison of HMTBA

  9. Amino acids

    MedlinePlus

    ... amino acids are: histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan , and valine. Nonessential amino acids "Nonessential" means that our bodies produce an amino ...

  10. Comparative analysis of some essential amino acids and available lysine in Acacia colei and A. tumida seeds using chemical methods and an amino acid analyzer.

    PubMed

    Falade, Olumuyiwa S; Adewusi, Steve R A

    2013-01-01

    Methionine, cysteine, tryptophan, and available lysine were determined in Acacia colei and A. tumida seeds and some cereals using chemical methods, and the results were compared to those obtained using an amino acid analyzer. Ba(OH)2 hydrolysis gave the best result of the three methods of hydrolysis (acid, base, and enzyme) tried. Oxidized methionine, cysteine, and tryptophan were not detected, but S-carboxyethylcysteine was estimated as cysteine by the chemical methods, thus overestimating cysteine's content in Acacia seeds. Tryptophan and methionine were higher in cereals than in Acacia seeds, while the level of cysteine and available lysine was higher in Acacia seeds than in cereals. These results agreed with values obtained using the amino acid analyzer and could therefore be used in low budget laboratories.

  11. S-adenosyl methionine is necessary for inhibition of the methyltransferase G9a by the lysine 9 to methionine mutation on histone H3

    PubMed Central

    Jayaram, Hariharan; Hoelper, Dominik; Jain, Siddhant U.; Cantone, Nico; Lundgren, Stefan M.; Poy, Florence; Allis, C. David; Cummings, Richard; Bellon, Steven; Lewis, Peter W.

    2016-01-01

    Lysine to methionine (K-to-M) mutations in genes encoding histone H3 are thought to drive a subset of pediatric brain and bone cancers. These high-frequency K-to-M mutations occur at sites of methylation on histone H3, and tumors containing the mutant histones exhibit a global loss of specific histone methylation marks. Previous studies showed that K-to-M mutant histones, also known as oncohistones, are potent orthosteric inhibitors of specific Su(var)3-9, Enhancer-of-zeste, Trithorax (SET) domain methyltransferases. However, the biochemical and biophysical details of the interaction between K-to-M mutant histones and the respective SET domain methyltransferases are currently unknown. Here, we use the histone H3K9-directed methyltransferase G9a as a model to explore the mechanism of inhibition by K-to-M oncohistones. X-ray cocrystal structures revealed that the K9M residue of histone H3 occupies the active site cavity of G9a, and kinetic analysis indicates competitive inhibition of G9a by histone H3K9M. Additionally, we find that the cofactor S-adenosyl methionine (SAM) is necessary for stable interaction between G9a and H3K9M histone. Consistent with the formation of a ternary complex, we find that the inhibitory peptide is uncompetitive with regard to SAM. These data and others indicate that K-to-M oncohistones promote global loss of specific lysine methylation through sequestration and inhibition of SAM-bound SET domain methyltransferases. PMID:27185940

  12. Nutritional availability of methionine, lysine and tryptophan in fish meals, as assessed with biological, microbiological and dye-binding assay procedures.

    PubMed

    Hewitt, D; Ford, J E

    1985-05-01

    In vitro assay procedures were applied in the measurement of available amino acids in a selection of fish meals representing good- and poor-quality product. Results were assessed by comparing them with results from chick-growth assays. Available methionine and tryptophan were assayed microbiologically with Streptococcus zymogenes, after predigestion of the test samples with papain or pronase. Results for methionine were correlated with chick-growth assays (r 0.86 for papain, 0.91 for pronase; P less than 0.01). Compared with the chick assays, papain tended to give lower, and pronase higher, results. Finer milling of the test samples did not influence the pronase values. Results for available tryptophan were also correlated with chick-growth assays (r 0.95 for papain, 0.96 for pronase; P less than 0.001). Compared with the chick values, papain gave markedly lower results and pronase marginally higher ones. Finer milling of the test samples increased the papain values by about 50% but had no effect with pronase. Available lysine was assayed microbiologically with Tetrahymena pyriformis and with a dye-binding procedure (DBL). The results correlated with the chick-growth assays (r 0.99 for DBL, P less than 0.001; 0.85 for Tetrahymena, P less than 0.01) but both methods overrated the poorer-quality samples. True nitrogen digestibilities and amino acid digestibilities were determined with chickens by the 'ileal analysis' procedure: the amino acid digestibilities were significantly higher and similar to the corresponding availabilities as measured in chick-growth assays. Ball milling a poor-quality fish meal caused a marked fall in its N digestibility, whereas similar treatment of a good-quality meal caused a slight increase. An explanation for this finding is proposed. Strep. zymogenes assays following pronase digestion of the test samples gave precise and acceptably accurate measures of the biologically available methionine and tryptophan in the test samples. For

  13. Effects of castration age, dietary protein level and lysine/methionine ratio on animal performance, carcass and meat quality of Friesian steers intensively reared.

    PubMed

    Prado, I N; Campo, M M; Muela, E; Valero, M V; Catalan, O; Olleta, J L; Sañudo, C

    2014-09-01

    The effects of castration age, dietary protein level and the dietary lysine/methionine (lys/met) ratio on animal performance, carcass characteristics and meat quality were studied in 64 intensively reared Friesian steers. Animals underwent castration procedures at 15 days old or at 5 months old. Dietary treatments started at 90 days old, with eight animals from each castration age randomly allocated to each treatment: 14.6% v. 16.8% CP (DM basis), and 3.0 v. 3.4 lys/met, on a 2×2×2 design. The recommended ratio of 3.0 was reached with supplementation of protected methionine. Steers were slaughtered at 443.5±26.2 kg live weight when they reached 12 months old approximately. Average daily gain, cold carcass weight or carcass classification were not affected by any studied effect. Muscle moisture (P=0.024), C18:2n-6 percentage (P=0.047), polyunsaturated fatty acid/saturated fatty acid (P=0.049) and n-6/n-3 (P=0.003) were higher in late castrated animals. Both high levels of dietary protein (P=0.008) and lys/met ratio (P=0.048) increased the percentage of muscle in the carcass. A level of 16.8% of CP in the diet also increased the percentage of monounsaturated fatty acids in the intramuscular fat (P=0.032), whereas a ratio lys/met of 3.4 decreased the percentage of saturated fatty acids (P=0.028). Thus, it is recommended using diets with a high protein level (16.8%) and a high lys/met ratio (3.4) in animals slaughtered at a young age, in order to obtain carcasses with high muscle content without negatively affecting productive traits or intramuscular fat composition.

  14. Lysine biotinylation and methionine oxidation in the heat shock protein HSP60 synergize in the elimination of reactive oxygen species in human cell cultures†

    PubMed Central

    Li, Yong; Malkaram, Sridhar A; Zhou, Jie; Zempleni, Janos

    2014-01-01

    Previous studies suggest that the number of proteins containing covalently bound biotin is larger than previously thought. Here, we report the identity of some of these proteins. Using mass spectrometry we discovered 108 novel biotinylation sites in the human embryonic kidney HEK293 cell proteome; members of the heat shock protein (HSP) superfamily were overrepresented among the novel biotinylated proteins. About half of the biotinylated proteins also displayed various degrees of methionine oxidation, which is known to play an important role in the defense against reactive oxygen species; for biotinylated HSPs, the percent of methionine sulfoxidation approached 100%. Protein structure analysis suggests that methionine sulfoxides localize in close physical proximity to the biotinylated lysines on the protein surface. Mass spectrometric analysis revealed that between 1 and 5 of the methionine residues in the C-terminal KEEKDPGMGAMGGMGGGMGGGMF motif are oxidized in HSP60. The likelihood of methionine sulfoxidation is higher if one of the adjacent lysine residues is biotinylated. Knockdown of HSP60 caused a 60% increase in the level of reactive oxygen species in fibroblasts cultured in biotin-sufficient medium. When HEK293 cells were transferred from biotin-sufficient medium to biotin-free medium, the level of reactive oxygen species increased by >9 times compared with baseline controls and a time-response relationship was evident. High levels of methionine sulfoxidation coincided with cell cycle arrest in the G0/G1 and S phases in biotin-depleted cells. We conclude that biotinylation of lysines synergizes with sulfoxidation of methionines in heat-shock proteins such as HSP60 in the defense against reactive oxygen species. PMID:24582286

  15. Effects of rumen-protected methionine, lysine, and histidine on lactation performance of dairy cows.

    PubMed

    Giallongo, F; Harper, M T; Oh, J; Lopes, J C; Lapierre, H; Patton, R A; Parys, C; Shinzato, I; Hristov, A N

    2016-06-01

    The objective of this study was to evaluate the effects of supplementing a metabolizable protein (MP)-deficient diet with rumen-protected (RP) Met, Lys, and His, individually or combined, on the performance of lactating dairy cows. The experiment was a 9-wk randomized complete block design with 72 Holstein cows. Following a 2-wk covariate period, cows were blocked by days in milk, milk yield, and parity, and randomly assigned to 1 of the following 6 treatments: (1) MP-adequate diet [MPA; +243g/d MP balance, according to the National Research Council (2001) requirements]; (2) MP-deficient diet (MPD; -54g/d MP balance); (3) MPD supplemented with RPMet (MPDM); (4) MPD supplemented with RPLys (MPDL); (5) MPD supplemented with RPHis (MPDH); and (6) MPD supplemented with RPMet, RPLys, and RPHis (MPDMLH). Dry matter intake (DMI), yields of milk and milk components (fat, protein, lactose) and energy-corrected milk (ECM), feed and ECM feed efficiencies, and milk and plasma urea N were decreased by MPD, compared with MPA. Supplementation of the MPD diet with RPLys increased milk protein content and plasma glucose concentration and tended to increase milk urea N. Addition of RPHis tended to increase DMI, increased milk protein concentration, and numerically increased yields of milk fat, protein, and ECM. In addition to the trends for increased DMI and milk fat content, and higher milk protein concentration, supplementation of the 3 RP AA also increased yields of milk fat, protein, and ECM and ECM feed efficiency. Relative to MPA, milk N efficiency tended to be increased by MPD. Concentrations of plasma essential AA (except Met and Thr) were decreased by MPD compared with MPA. Supplementation of RPMet, RPLys, and RPHis increased plasma Met (except for MPDM), Lys, and His concentrations, respectively. Cows fed MPD had lower blood hemoglobin concentration and numerically higher plasma ghrelin than cows fed MPA. Concentration of total saturated fatty acids in milk fat were or

  16. Effects of supplemental lysine and methionine with zilpaterol hydrochloride on feedlot performance, carcass merit, and skeletal muscle fiber characteristics in finishing feedlot cattle.

    PubMed

    Hosford, A D; Hergenreder, J E; Kim, J K; Baggerman, J O; Ribeiro, F R B; Anderson, M J; Spivey, K S; Rounds, W; Johnson, B J

    2015-09-01

    Feeding zilpaterol hydrochloride (ZH) with ruminally protected AA was evaluated in a small-pen feeding trial. Crossbred steers ( = 180; initial BW = 366 kg) were blocked by weight and then randomly assigned to treatments (45 pens; 9 pens/treatment). Treatment groups consisted of no ZH and no AA (Cont-), ZH and no AA (Cont+), ZH and a ruminally protected lysine supplement (Lys), ZH and a ruminally protected methionine supplement (Met), and ZH and ruminally protected lysine and methionine (Lys+Met). Zilpaterol hydrochloride (8.3 mg/kg DM) was fed for the last 20 d of the finishing period with a 3-d withdrawal period. Lysine and Met were top dressed daily for the 134-d feeding trial to provide 12 or 4 g·hd·d, respectively, to the small intestine. Carcass characteristics, striploins, and prerigor muscle samples were collected following harvest at a commercial facility. Steaks from each steer were aged for 7, 14, 21, and 28 d, and Warner-Bratzler shear force (WBSF) was determined as an indicator of tenderness. Prerigor muscle samples were used for immunohistological analysis. Cattle treated with Met and Lys+Met had increased final BW ( < 0.3) and ADG ( < 0.05) compared to Cont- and Cont+. Supplementation of Lys, Met, and Lys+Met improved G:F ( < 0.05) compared to Cont- during the ZH feeding period (d 111 to 134) as well as the entire feeding period ( < 0.05). Zilpaterol hydrochloride increased carcass ADG ( < 0.05) when compared to non-ZH-fed steers. Methionine and Lys+Met treatments had heavier HCW ( < 0.02) than that of Cont-. Yield grade was decreased ( < 0.04) for Cont+ steers compared to steers treated with Lys, Lys+Met, and Cont-. Tenderness was reduced ( < 0.05) with ZH regardless of AA supplementation. Lysine, Met, Lys+Met, and Cont+ had less tender steaks ( < 0.05) throughout all aging groups compared to Cont-. Steaks from Lys-treated steers were less tender ( < 0.05) than those of Cont+ during the 7- and 14-d aging periods. Nuclei density was the greatest

  17. Histidine-lysine peptides as carriers of nucleic acids.

    PubMed

    Leng, Qixin; Goldgeier, Lisa; Zhu, Jingsong; Cambell, Patricia; Ambulos, Nicholas; Mixson, A James

    2007-03-01

    With their biodegradability and diversity of permutations, peptides have significant potential as carriers of nucleic acids. This review will focus on the sequence and branching patterns of peptide carriers composed primarily of histidines and lysines. While lysines within peptides are important for binding to the negatively charged phosphates, histidines are critical for endosomal lysis enabling nucleic acids to reach the cytosol. Histidine-lysine (HK) polymers by either covalent or ionic bonds with liposomes augment transfection compared to liposome carriers alone. More recently, we have examined peptides as sole carriers of nucleic acids because of their intrinsic advantages compared to the bipartite HK/liposome carriers. With a protocol change and addition of a histidine-rich tail, HK peptides as sole carriers were more effective than liposomes alone in several cell lines. While four-branched polymers with a primary repeating sequence pattern of -HHK- were more effective as carriers of plasmids, eight-branched polymers with a sequence pattern of -HHHK- were more effective as carriers of siRNA. Compared to polyethylenimine, HK carriers of siRNA and plasmids had reduced toxicity. When injected intravenously, HK polymers in complex with plasmids encoding antiangiogenic proteins significantly decreased tumor growth. Furthermore, modification of HK polymers with polyethylene glycol and vascular-specific ligands increased specificity of the polyplex to the tumor by more than 40-fold. Together with further development and insight on the structure of HK polyplexes, HK peptides may prove to be useful as carriers of different forms of nucleic acids both in vitro and in vivo. PMID:17440630

  18. Effects of dietary lysine and methionine supplementation on ross 708 male broilers from 21 to 42 days of age (III): serum metabolites, hormones, and their relationship with growth performance

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A previous study has shown that a limited increase of lysine (Lys) and methionine (Met) in broiler diets may improve feed conversion ratio (FCR), BW, carcass yield, and breast meat yield. This study was conducted to determine the effects of dietary Lys and Met supplementation on various blood serum ...

  19. Efficacy of DL-methionine hydroxy analogue-free acid in comparison to DL-methionine in growing male white Pekin ducks.

    PubMed

    Kluge, H; Gessner, D K; Herzog, E; Eder, K

    2016-03-01

    The present study was performed to assess the bioefficacy of DL-methionine hydroxy analogue-free acid (MHA) in comparison to DL-methionine (DLM) as sources of methionine for growing male white Pekin ducks in the first 3 wk of life. For this aim, 580 1-day-old male ducks were allocated into 12 treatment groups and received a basal diet that contained 0.29% of methionine, 0.34% of cysteine and 0.63% of total sulphur containing amino acids or the same diet supplemented with either DLM or MHA in amounts to supply 0.05, 0.10, 0.15, 0.20, and 0.25% of methionine equivalents. Ducks fed the control diet without methionine supplement had the lowest final body weights, daily body weight gains and feed intake among all groups. Supplementation of methionine improved final body weights and daily body weight gains in a dose dependent-manner. There was, however, no significant effect of the source of methionine on all of the performance responses. Evaluation of the data of daily body weight gains with an exponential model of regression revealed a nearly identical efficacy (slope of the curves) of both compounds for growth (DLM = 100%, MHA = 101%). According to the exponential model of regression, 95% of the maximum values of daily body weight gain were reached at methionine supplementary levels of 0.080% and 0.079% for DLM and MHA, respectively. Overall, the present study indicates that MHA and DLM have a similar efficacy as sources of methionine for growing ducks. It is moreover shown that dietary methionine concentrations of 0.37% are required to reach 95% of the maximum of daily body weight gains in ducks during the first 3 wk of life.

  20. 21 CFR 172.320 - Amino acids.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ...-Leucine L-Lysine DL-Methionine (not for infant foods) L-Methionine L-Phenylalanine L-Proline L-Serine L... Aminoacetic acid (glycine) L-Leucine DL-Methionine L-Methionine L-Tryptophan L-Phenylalanine L-Proline L... DL-Methionine 3.1 L-Phenylalanine 5.8 L-Proline 4.2 L-Serine 8.4 L-Threonine 5.0 L-Tryptophan 1.6...

  1. 21 CFR 172.320 - Amino acids.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ...-Leucine L-Lysine DL-Methionine (not for infant foods) L-Methionine L-Phenylalanine L-Proline L-Serine L... Aminoacetic acid (glycine) L-Leucine DL-Methionine L-Methionine L-Tryptophan L-Phenylalanine L-Proline L... DL-Methionine 3.1 L-Phenylalanine 5.8 L-Proline 4.2 L-Serine 8.4 L-Threonine 5.0 L-Tryptophan 1.6...

  2. 21 CFR 172.320 - Amino acids.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ...-Leucine L-Lysine DL-Methionine (not for infant foods) L-Methionine L-Phenylalanine L-Proline L-Serine L... Aminoacetic acid (glycine) L-Leucine DL-Methionine L-Methionine L-Tryptophan L-Phenylalanine L-Proline L... DL-Methionine 3.1 L-Phenylalanine 5.8 L-Proline 4.2 L-Serine 8.4 L-Threonine 5.0 L-Tryptophan 1.6...

  3. Safety, efficacy and physiological actions of a lysine-free, arginine-rich formula to treat glutaryl-CoA dehydrogenase deficiency: focus on cerebral amino acid influx.

    PubMed

    Strauss, Kevin A; Brumbaugh, Joan; Duffy, Alana; Wardley, Bridget; Robinson, Donna; Hendrickson, Christine; Tortorelli, Silvia; Moser, Ann B; Puffenberger, Erik G; Rider, Nicholas L; Morton, D Holmes

    2011-01-01

    Striatal degeneration from glutaryl-CoA dehydrogenase deficiency (glutaric aciduria type 1, GA1) is associated with cerebral formation and entrapment of glutaryl-CoA and its derivatives that depend on cerebral lysine influx. In 2006 we designed a lysine-free study formula enriched with arginine to selectively block lysine transport across cerebral endothelia and thereby limit glutaryl-CoA production by brain. Between 2006 and present, we treated twelve consecutive children with study formula (LYSx group) while holding all other treatment practices constant. Clinical and biochemical outcomes were compared to 25 GA1 patients (PROx group) treated between 1995 and 2005 with natural protein restriction (dietary lysine/arginine ratio of 1.7±0.3 mg:mg). We used published kinetic parameters of the y+and LAT1 blood-brain barrier transporters to model the influx of amino acids into the brain. Arginine fortification to achieve a mean dietary lysine/arginine ratio of 0.7±0.2 mg:mg was neuroprotective. All 12 LYSx patients are physically and neurologically healthy after 28 aggregate patient-years of follow up (current ages 28±21 months) and there were no adverse events related to formula use. This represents a 36% reduction of neurological risk (95% confidence interval 14-52%, p=0.018) that we can directly attribute to altered amino acid intake. During the first year of life, 20% lower lysine intake and two-fold higher arginine intake by LYSx patients were associated with 50% lower plasma lysine, 3-fold lower plasma lysine/arginine concentration ratio, 42% lower mean calculated cerebral lysine influx, 54% higher calculated cerebral arginine influx, 15-26% higher calculated cerebral influx of several anaplerotic precursors (isoleucine, threonine, methionine, and leucine), 50% less 3-hydroxyglutarate excretion, and a 3-fold lower hospitalization rate (0.8 versus 2.3 hospitalizations per patient per year). The relationship between arginine fortification and plasma lysine

  4. Pathways of Amino Acid Degradation in Nilaparvata lugens (Stål) with Special Reference to Lysine-Ketoglutarate Reductase/Saccharopine Dehydrogenase (LKR/SDH)

    PubMed Central

    Wan, Pin-Jun; Yuan, San-Yue; Tang, Yao-Hua; Li, Kai-Long; Yang, Lu; Fu, Qiang; Li, Guo-Qing

    2015-01-01

    Nilaparvata lugens harbors yeast-like symbionts (YLSs). In present paper, a genome-wide analysis found 115 genes from Ni. lugens and 90 genes from YLSs that were involved in the metabolic degradation of 20 proteinogenic amino acids. These 205 genes encoded for 77 enzymes. Accordingly, the degradation pathways for the 20 amino acids were manually constructed. It is postulated that Ni. lugens can independently degrade fourteen amino acids (threonine, alanine, glycine, serine, aspartate, asparagine, phenylalanine, tyrosine, glutamate, glutamine, proline, histidine, leucine and lysine). Ni. lugens and YLSs enzymes may work collaboratively to break down tryptophan, cysteine, arginine, isoleucine, methionine and valine. We cloned a lysine-ketoglutarate reductase/saccharopine dehydrogenase gene (Nllkr/sdh) that encoded a bifunctional enzyme catalyzing the first two steps of lysine catabolism. Nllkr/sdh is widely expressed in the first through fifth instar nymphs and adults, and is highly expressed in the fat body, ovary and gut in adults. Ingestion of dsNllkr/sdh by nymphs successfully knocked down the target gene, and caused nymphal/adult mortality, shortened nymphal development stage and reduced adult fresh weight. Moreover, Nllkr/sdh knockdown resulted in three defects: wings were shortened and thickened; cuticles were stretched and thinned; and old nymphal cuticles remained on the tips of legs and abdomen and were not completely shed. These data indicate that impaired lysine degradation negatively affects the survival and development of Ni. lugens. PMID:26000452

  5. Effects of castration age, protein level and lysine/methionine ratio in the diet on colour, lipid oxidation and meat acceptability of intensively reared Friesian steers.

    PubMed

    Prado, I N; Campo, M M; Muela, E; Valero, M V; Catalan, O; Olleta, J L; Sañudo, C

    2015-08-01

    A total of 64 intensively reared Friesian steers were used in a 2×2×2 design to study the effects of age of castration (15 days old v. 5 months old), dietary protein level (14.6% v. 16.8%; DM basis) and lysine/methionine (lys/met) ratio (3.0 v. 3.4) on meat quality. The lys/met ratio of 3.0 was reached with supplementation of protected methionine. Animals were slaughtered at a live weight of 443.5 ± 26.2 kg at around 12 months of age. Colour and lipid oxidation were measured in the longissimus thoracis muscle throughout the 14 days of display under modified atmospheric and commercial display conditions. A panel of 17 consumers assessed daily the visual acceptability of the meat on display. A consumer acceptability eating test was also performed with 120 consumers in meat aged for 7 days under vacuum conditions. Lipid oxidation was not influenced by castration age and the protein level in the diet. Castration age did not affect meat colour, but meat from the low protein level diet and the low lys/met ratio showed higher redness (a*) from 3 days of display onwards. Nevertheless, from 6 days onwards, consumer visual acceptability was below the level of acceptance in all treatments, and even from 5 days onwards in those animals that underwent early castration and were fed either a high protein diet or a combination diet low in protein content and high in lys/met ratio. The best accepted treatments throughout the display period were those from late castrated animals fed a low protein diet, probably related to other visual aspects. However, the best accepted meat after consumption was that from late castrated animals fed high protein and high lys/met. The addition of protected methionine to reach lys/met levels of 3.0 did not improve beef acceptability, with the high protein diet being preferred by consumers in terms of palatability in late castrated animals. PMID:26190253

  6. Effects of castration age, protein level and lysine/methionine ratio in the diet on colour, lipid oxidation and meat acceptability of intensively reared Friesian steers.

    PubMed

    Prado, I N; Campo, M M; Muela, E; Valero, M V; Catalan, O; Olleta, J L; Sañudo, C

    2015-08-01

    A total of 64 intensively reared Friesian steers were used in a 2×2×2 design to study the effects of age of castration (15 days old v. 5 months old), dietary protein level (14.6% v. 16.8%; DM basis) and lysine/methionine (lys/met) ratio (3.0 v. 3.4) on meat quality. The lys/met ratio of 3.0 was reached with supplementation of protected methionine. Animals were slaughtered at a live weight of 443.5 ± 26.2 kg at around 12 months of age. Colour and lipid oxidation were measured in the longissimus thoracis muscle throughout the 14 days of display under modified atmospheric and commercial display conditions. A panel of 17 consumers assessed daily the visual acceptability of the meat on display. A consumer acceptability eating test was also performed with 120 consumers in meat aged for 7 days under vacuum conditions. Lipid oxidation was not influenced by castration age and the protein level in the diet. Castration age did not affect meat colour, but meat from the low protein level diet and the low lys/met ratio showed higher redness (a*) from 3 days of display onwards. Nevertheless, from 6 days onwards, consumer visual acceptability was below the level of acceptance in all treatments, and even from 5 days onwards in those animals that underwent early castration and were fed either a high protein diet or a combination diet low in protein content and high in lys/met ratio. The best accepted treatments throughout the display period were those from late castrated animals fed a low protein diet, probably related to other visual aspects. However, the best accepted meat after consumption was that from late castrated animals fed high protein and high lys/met. The addition of protected methionine to reach lys/met levels of 3.0 did not improve beef acceptability, with the high protein diet being preferred by consumers in terms of palatability in late castrated animals.

  7. Evolution of initiator tRNAs and selection of methionine as the initiating amino acid.

    PubMed

    Bhattacharyya, Souvik; Varshney, Umesh

    2016-09-01

    Transfer RNAs (tRNAs) have been important in shaping biomolecular evolution. Initiator tRNAs (tRNAi), a special class of tRNAs, carry methionine (or its derivative, formyl-methionine) to ribosomes to start an enormously energy consuming but a highly regulated process of protein synthesis. The processes of tRNAi evolution, and selection of methionine as the universal initiating amino acid remain an enigmatic problem. We constructed phylogenetic trees using the whole sequence, the acceptor-TψC arm ('minihelix'), and the anticodon-dihydrouridine arm regions of tRNAi from 158 species belonging to all 3 domains of life. All the trees distinctly assembled into 3 domains of life. Large trees, generated using data for all the tRNAs of a vast number of species, fail to reveal the major evolutionary events and identity of the probable elongator tRNA sequences that could be ancestor of tRNAi. Therefore, we constructed trees using the minihelix or the whole sequence of species specific tRNAs, and iterated our analysis on 50 eubacterial species. We identified tRNA(Pro), tRNA(Glu), or tRNA(Thr) (but surprisingly not elongator tRNA(Met)) as probable ancestors of tRNAi. We then determined the factors imposing selection of methionine as the initiating amino acid. Overall frequency of occurrence of methionine, whose metabolic cost of synthesis is the highest among all amino acids, remains almost unchanged across the 3 domains of life. Our correlation analysis shows that its high metabolic cost is independent of many physicochemical properties of the side chain. Our results indicate that selection of methionine, as the initiating amino acid was possibly a consequence of the evolution of one-carbon metabolism, which plays an important role in regulating translation initiation. PMID:27322343

  8. Voluntary wheel running is beneficial to the amino acid profile of lysine-deficient rats.

    PubMed

    Nagao, Kenji; Bannai, Makoto; Seki, Shinobu; Kawai, Nobuhiro; Mori, Masato; Takahashi, Michio

    2010-06-01

    Rats voluntarily run up to a dozen kilometers per night when their cages are equipped with a running wheel. Daily voluntary running is generally thought to enhance protein turnover. Thus, we sought to determine whether running worsens or improves protein degradation caused by a lysine-deficient diet and whether it changes the utilization of free amino acids released by proteolysis. Rats were fed a lysine-deficient diet and were given free access to a running wheel or remained sedentary (control) for 4 wk. Amino acid levels in plasma, muscle, and liver were measured together with plasma insulin levels and tissue weight. The lysine-deficient diet induced anorexia, skeletal muscle loss, and serine and threonine aminoacidemia, and it depleted plasma insulin and essential amino acids in skeletal muscle. Allowing rats to run voluntarily improved these symptoms; thus, voluntary wheel running made the rats less susceptible to dietary lysine deficiency. Amelioration of the declines in muscular leucine and plasma insulin observed in running rats could contribute to protein synthesis together with the enhanced availability of lysine and other essential amino acids in skeletal muscle. These results indicate that voluntary wheel running under lysine-deficient conditions does not enhance protein catabolism; on the contrary, it accelerates protein synthesis and contributes to the maintenance of muscle mass. The intense nocturnal voluntary running that characterizes rodents might be an adaptation of lysine-deficient grain eaters that allows them to maximize opportunities for food acquisition. PMID:20233939

  9. Structural Insights Into Amino Acid Binding and Gene Control by a Lysine Riboswitch

    SciTech Connect

    Serganov, A.; Huang, L; Patel, D

    2008-01-01

    In bacteria, the intracellular concentration of several amino acids is controlled by riboswitches1, 2, 3, 4. One of the important regulatory circuits involves lysine-specific riboswitches, which direct the biosynthesis and transport of lysine and precursors common for lysine and other amino acids. To understand the molecular basis of amino acid recognition by riboswitches, here we present the crystal structure of the 174-nucleotide sensing domain of the Thermotoga maritima lysine riboswitch in the lysine-bound (1.9 A) and free (3.1 A) states. The riboswitch features an unusual and intricate architecture, involving three-helical and two-helical bundles connected by a compact five-helical junction and stabilized by various long-range tertiary interactions. Lysine interacts with the junctional core of the riboswitch and is specifically recognized through shape-complementarity within the elongated binding pocket and through several direct and K+-mediated hydrogen bonds to its charged ends. Our structural and biochemical studies indicate preformation of the riboswitch scaffold and identify conformational changes associated with the formation of a stable lysine-bound state, which prevents alternative folding of the riboswitch and facilitates formation of downstream regulatory elements. We have also determined several structures of the riboswitch bound to different lysine analogues5, including antibiotics, in an effort to understand the ligand-binding capabilities of the lysine riboswitch and understand the nature of antibiotic resistance. Our results provide insights into a mechanism of lysine-riboswitch-dependent gene control at the molecular level, thereby contributing to continuing efforts at exploration of the pharmaceutical and biotechnological potential of riboswitches.

  10. A Candida guilliermondii lysine hyperproducer capable of elevated citric acid production.

    PubMed

    West, Thomas P

    2016-05-01

    A mutant of the yeast Candida guilliermondii ATCC 9058 exhibiting elevated citric acid production was isolated based upon its ability to overproduce lysine. This method involved the use of a solid medium containing a combination of lysine analogues to identify a mutant that produced a several-fold higher lysine level compared to its parent strain using glucose or glycerol as a carbon source. The mutant strain was also capable of producing more than a fivefold higher citric acid level on glycerol as a carbon source compared to its parent strain. It was concluded that the screening of yeast lysine hyperproducer strains could provide a rapid approach to isolate yeast citric acid hyperproducer strains. PMID:27038943

  11. A Candida guilliermondii lysine hyperproducer capable of elevated citric acid production.

    PubMed

    West, Thomas P

    2016-05-01

    A mutant of the yeast Candida guilliermondii ATCC 9058 exhibiting elevated citric acid production was isolated based upon its ability to overproduce lysine. This method involved the use of a solid medium containing a combination of lysine analogues to identify a mutant that produced a several-fold higher lysine level compared to its parent strain using glucose or glycerol as a carbon source. The mutant strain was also capable of producing more than a fivefold higher citric acid level on glycerol as a carbon source compared to its parent strain. It was concluded that the screening of yeast lysine hyperproducer strains could provide a rapid approach to isolate yeast citric acid hyperproducer strains.

  12. Biofortification of rice with the essential amino acid lysine: molecular characterization, nutritional evaluation, and field performance.

    PubMed

    Yang, Qing-Qing; Zhang, Chang-Quan; Chan, Man-Ling; Zhao, Dong-Sheng; Chen, Jin-Zhu; Wang, Qing; Li, Qian-Feng; Yu, Heng-Xiu; Gu, Ming-Hong; Sun, Samuel Sai-Ming; Liu, Qiao-Quan

    2016-07-01

    Rice (Oryza sativa L.), a major staple crop worldwide, has limited levels of the essential amino acid lysine. We previously produced engineered rice with increased lysine content by expressing bacterial aspartate kinase and dihydrodipicolinate synthase and inhibiting rice lysine ketoglutarate reductase/saccharopine dehydrogenase activity. However, the grain quality, field performance, and integration patterns of the transgenes in these lysine-enriched lines remain unclear. In the present study, we selected several elite transgenic lines with endosperm-specific or constitutive regulation of the above key enzymes but lacking the selectable marker gene. All target transgenes were integrated into the intragenic region in the rice genome. Two pyramid transgenic lines (High Free Lysine; HFL1 and HFL2) with free lysine levels in seeds up to 25-fold that of wild type were obtained via a combination of the above two transgenic events. We observed a dramatic increase in total free amino acids and a slight increase in total protein content in both pyramid lines. Moreover, the general physicochemical properties were improved in pyramid transgenic rice, but the starch composition was not affected. Field trials indicated that the growth of HFL transgenic rice was normal, except for a slight difference in plant height and grain colour. Taken together, these findings will be useful for the potential commercialization of high-lysine transgenic rice. PMID:27252467

  13. Impact of food supplementation and methionine on high densities of cotton rats: Support of the amino-acid-quality hypothesis?

    USGS Publications Warehouse

    Webb, R.E.; Leslie, David M.; Lochmiller, R.L.; Masters, R.E.

    2005-01-01

    Considerable research supports the tenet that quantity and quality of food limit vertebrate populations. We evaluated predictions that increased availabilities of food and the essential amino acid methionine were related to population limitation of the hispid cotton rat (Sigmodon hispidus). Effects of supplemental food and methionine on density, survival, and reproductive parameters of wild cotton rats were assessed in north-central Oklahoma in 1998-1999. Twelve enclosed groups of 16 adult cotton rats each (8 male, 8 female) were randomly assigned to either no supplementation (control), supplementation with a mixed ration that had methionine at slightly below maintenance levels (0.20%), or a methionine-enhanced mixed ration (1.20%). In general, densities of cotton rats were twice as high and were sustained longer with dietary supplementation, and methionine-supplemented populations maintained the highest densities. Treatment effects on survival depended on time of year, with higher survival in supplemented enclosures in October and November. Per capita recruitment was highest with methionine-enhanced food. Treatment effects on proportions of overall and female cotton rats in reproductive condition depended on sampling date, but males were most reproductively active with methionine supplementation. Methionine supplementation resulted in an earlier and longer reproductive season. Density-dependent and density-independent factors no doubt interplay to determine population dynamics of cotton rats, but our results suggest that methionine plays a role in the population dynamics of wild cotton rats, apparently by enhancing overall density, recruitment, and reproductive activity of males.

  14. Interaction studies of human prion protein (HuPrP109-111: methionine-lysine-histidine) tripeptide model with transition metal cations.

    PubMed

    Pitchumani Violet Mary, C; Shankar, R; Vijayakumar, S; Kolandaivel, P

    2016-09-01

    In the present study, the coordination bonds between the Methionine-Lysine-Histidine (Ac-MKH-NHMe) tripeptide model associated with the fifth metal binding site, which triggers the β-sheet formation of human prion protein and the divalent metal cations such as Mn(2+), Cu(2+) and Zn(2+) were studied using B3LYP and M052X levels of theory with LANL2DZ basis set. For each transition divalent metal cation, three different coordination modes (4N, 3NO, and 2NSO) were analyzed. The present result reveals that overall structural parameters of MKH model tripeptide are altered due to the interaction of divalent metal cations. Among these three coordination modes, the 4N-M(2)(+) and 4N2O-Mn(2+) complexes are found to have the larger interaction energy, MIA and deformation energies. The triply deprotonated coordination mode of the Ac-MKH-NHMe tripeptide transfers more amount of charge to the divalent metal cations than the dually and singly deprotonated complexes. Furthermore, the atoms in molecules (AIM) topological analysis confirm that, the interaction between the metal cations Mn(2+), Cu(2+) and Zn(2+) and Ac-MKH-NHMe tripeptide are electrostatic dominant and the coordination modes with triply deprotonation states possess larger electron density at their BCP corresponding to their coordination bonds. The electrostatic potential difference maps of the most stable 4N-M(2+) (M(2+)=Cu(2+) and Zn(2+)) and 4N2O-Mn(2+) reveals that, as the ionic radii of the metal ion increases, the delocalization charges localized on the metal cations are found to be decreased. The Infra-red stretching frequencies of NH, CH, and CH2 groups of each coordination complexes are observed with shift in their stretching frequencies. From these observations we conclude that, the transition divalent metal cations binding in 4N coordination mode will induce more conformational changes of the Prion protein.

  15. Interaction studies of human prion protein (HuPrP109-111: methionine-lysine-histidine) tripeptide model with transition metal cations.

    PubMed

    Pitchumani Violet Mary, C; Shankar, R; Vijayakumar, S; Kolandaivel, P

    2016-09-01

    In the present study, the coordination bonds between the Methionine-Lysine-Histidine (Ac-MKH-NHMe) tripeptide model associated with the fifth metal binding site, which triggers the β-sheet formation of human prion protein and the divalent metal cations such as Mn(2+), Cu(2+) and Zn(2+) were studied using B3LYP and M052X levels of theory with LANL2DZ basis set. For each transition divalent metal cation, three different coordination modes (4N, 3NO, and 2NSO) were analyzed. The present result reveals that overall structural parameters of MKH model tripeptide are altered due to the interaction of divalent metal cations. Among these three coordination modes, the 4N-M(2)(+) and 4N2O-Mn(2+) complexes are found to have the larger interaction energy, MIA and deformation energies. The triply deprotonated coordination mode of the Ac-MKH-NHMe tripeptide transfers more amount of charge to the divalent metal cations than the dually and singly deprotonated complexes. Furthermore, the atoms in molecules (AIM) topological analysis confirm that, the interaction between the metal cations Mn(2+), Cu(2+) and Zn(2+) and Ac-MKH-NHMe tripeptide are electrostatic dominant and the coordination modes with triply deprotonation states possess larger electron density at their BCP corresponding to their coordination bonds. The electrostatic potential difference maps of the most stable 4N-M(2+) (M(2+)=Cu(2+) and Zn(2+)) and 4N2O-Mn(2+) reveals that, as the ionic radii of the metal ion increases, the delocalization charges localized on the metal cations are found to be decreased. The Infra-red stretching frequencies of NH, CH, and CH2 groups of each coordination complexes are observed with shift in their stretching frequencies. From these observations we conclude that, the transition divalent metal cations binding in 4N coordination mode will induce more conformational changes of the Prion protein. PMID:27611644

  16. Comprehensive Profiling of Amino Acid Response Uncovers Unique Methionine-Deprived Response Dependent on Intact Creatine Biosynthesis

    PubMed Central

    Tang, Xiaohu; Keenan, Melissa M.; Wu, Jianli; Lin, Chih-An; Dubois, Laura; Thompson, J. Will; Freedland, Stephen J.; Murphy, Susan K.; Chi, Jen-Tsan

    2015-01-01

    Besides being building blocks for protein synthesis, amino acids serve a wide variety of cellular functions, including acting as metabolic intermediates for ATP generation and for redox homeostasis. Upon amino acid deprivation, free uncharged tRNAs trigger GCN2-ATF4 to mediate the well-characterized transcriptional amino acid response (AAR). However, it is not clear whether the deprivation of different individual amino acids triggers identical or distinct AARs. Here, we characterized the global transcriptional response upon deprivation of one amino acid at a time. With the exception of glycine, which was not required for the proliferation of MCF7 cells, we found that the deprivation of most amino acids triggered a shared transcriptional response that included the activation of ATF4, p53 and TXNIP. However, there was also significant heterogeneity among different individual AARs. The most dramatic transcriptional response was triggered by methionine deprivation, which activated an extensive and unique response in different cell types. We uncovered that the specific methionine-deprived transcriptional response required creatine biosynthesis. This dependency on creatine biosynthesis was caused by the consumption of S-Adenosyl-L-methionine (SAM) during creatine biosynthesis that helps to deplete SAM under methionine deprivation and reduces histone methylations. As such, the simultaneous deprivation of methionine and sources of creatine biosynthesis (either arginine or glycine) abolished the reduction of histone methylation and the methionine-specific transcriptional response. Arginine-derived ornithine was also required for the complete induction of the methionine-deprived specific gene response. Collectively, our data identify a previously unknown set of heterogeneous amino acid responses and reveal a distinct methionine-deprived transcriptional response that results from the crosstalk of arginine, glycine and methionine metabolism via arginine

  17. Comprehensive profiling of amino acid response uncovers unique methionine-deprived response dependent on intact creatine biosynthesis.

    PubMed

    Tang, Xiaohu; Keenan, Melissa M; Wu, Jianli; Lin, Chih-An; Dubois, Laura; Thompson, J Will; Freedland, Stephen J; Murphy, Susan K; Chi, Jen-Tsan

    2015-04-01

    Besides being building blocks for protein synthesis, amino acids serve a wide variety of cellular functions, including acting as metabolic intermediates for ATP generation and for redox homeostasis. Upon amino acid deprivation, free uncharged tRNAs trigger GCN2-ATF4 to mediate the well-characterized transcriptional amino acid response (AAR). However, it is not clear whether the deprivation of different individual amino acids triggers identical or distinct AARs. Here, we characterized the global transcriptional response upon deprivation of one amino acid at a time. With the exception of glycine, which was not required for the proliferation of MCF7 cells, we found that the deprivation of most amino acids triggered a shared transcriptional response that included the activation of ATF4, p53 and TXNIP. However, there was also significant heterogeneity among different individual AARs. The most dramatic transcriptional response was triggered by methionine deprivation, which activated an extensive and unique response in different cell types. We uncovered that the specific methionine-deprived transcriptional response required creatine biosynthesis. This dependency on creatine biosynthesis was caused by the consumption of S-Adenosyl-L-methionine (SAM) during creatine biosynthesis that helps to deplete SAM under methionine deprivation and reduces histone methylations. As such, the simultaneous deprivation of methionine and sources of creatine biosynthesis (either arginine or glycine) abolished the reduction of histone methylation and the methionine-specific transcriptional response. Arginine-derived ornithine was also required for the complete induction of the methionine-deprived specific gene response. Collectively, our data identify a previously unknown set of heterogeneous amino acid responses and reveal a distinct methionine-deprived transcriptional response that results from the crosstalk of arginine, glycine and methionine metabolism via arginine

  18. One-Step Biosynthesis of α-Keto-γ-Methylthiobutyric Acid from L-Methionine by an Escherichia coli Whole-Cell Biocatalyst Expressing an Engineered L-Amino Acid Deaminase from Proteus vulgaris

    PubMed Central

    Shin, Hyun-dong; Du, Guocheng; Wang, Miao; Liu, Long; Chen, Jian

    2014-01-01

    α-Keto-γ-methylthiobutyric acid (KMTB), a keto derivative of l-methionine, has great potential for use as an alternative to l-methionine in the poultry industry and as an anti-cancer drug. This study developed an environment friendly process for KMTB production from l-methionine by an Escherichia coli whole-cell biocatalyst expressing an engineered l-amino acid deaminase (l-AAD) from Proteus vulgaris. We first overexpressed the P. vulgaris l-AAD in E. coli BL21 (DE3) and further optimized the whole-cell transformation process. The maximal molar conversion ratio of l-methionine to KMTB was 71.2% (mol/mol) under the optimal conditions (70 g/L l-methionine, 20 g/L whole-cell biocatalyst, 5 mM CaCl2, 40°C, 50 mM Tris-HCl [pH 8.0]). Then, error-prone polymerase chain reaction was used to construct P. vulgaris l-AAD mutant libraries. Among approximately 104 mutants, two mutants bearing lysine 104 to arginine and alanine 337 to serine substitutions showed 82.2% and 80.8% molar conversion ratios, respectively. Furthermore, the combination of these mutations enhanced the catalytic activity and molar conversion ratio by 1.3-fold and up to 91.4% with a KMTB concentration of 63.6 g/L. Finally, the effect of immobilization on whole-cell transformation was examined, and the immobilized whole-cell biocatalyst with Ca2+ alginate increased reusability by 41.3% compared to that of free cell production. Compared with the traditional multi-step chemical synthesis, our one-step biocatalytic production of KMTB has an advantage in terms of environmental pollution and thus has great potential for industrial KMTB production. PMID:25531756

  19. One-step biosynthesis of α-keto-γ-methylthiobutyric acid from L-methionine by an Escherichia coli whole-cell biocatalyst expressing an engineered L-amino acid deaminase from Proteus vulgaris.

    PubMed

    Hossain, Gazi Sakir; Li, Jianghua; Shin, Hyun-dong; Du, Guocheng; Wang, Miao; Liu, Long; Chen, Jian

    2014-01-01

    α-Keto-γ-methylthiobutyric acid (KMTB), a keto derivative of l-methionine, has great potential for use as an alternative to l-methionine in the poultry industry and as an anti-cancer drug. This study developed an environment friendly process for KMTB production from l-methionine by an Escherichia coli whole-cell biocatalyst expressing an engineered l-amino acid deaminase (l-AAD) from Proteus vulgaris. We first overexpressed the P. vulgaris l-AAD in E. coli BL21 (DE3) and further optimized the whole-cell transformation process. The maximal molar conversion ratio of l-methionine to KMTB was 71.2% (mol/mol) under the optimal conditions (70 g/L l-methionine, 20 g/L whole-cell biocatalyst, 5 mM CaCl2, 40°C, 50 mM Tris-HCl [pH 8.0]). Then, error-prone polymerase chain reaction was used to construct P. vulgaris l-AAD mutant libraries. Among approximately 104 mutants, two mutants bearing lysine 104 to arginine and alanine 337 to serine substitutions showed 82.2% and 80.8% molar conversion ratios, respectively. Furthermore, the combination of these mutations enhanced the catalytic activity and molar conversion ratio by 1.3-fold and up to 91.4% with a KMTB concentration of 63.6 g/L. Finally, the effect of immobilization on whole-cell transformation was examined, and the immobilized whole-cell biocatalyst with Ca2+ alginate increased reusability by 41.3% compared to that of free cell production. Compared with the traditional multi-step chemical synthesis, our one-step biocatalytic production of KMTB has an advantage in terms of environmental pollution and thus has great potential for industrial KMTB production.

  20. AMPHOTERIC BEHAVIOR OF COMPLEX SYSTEMS : III. THE CONDUCTIVITY OF SULFANILIC ACID-LYSIN MIXTURES.

    PubMed

    Stearn, A E

    1927-01-20

    Conductivities of sulfanilic acid, lysin, and mixtures of the two were made over a wide pH range, the pH being adjusted by means of phosphate buffers. The actual conductivities of the sulfanilic acid, the lysin, and the mixture were calculated. The difference between the conductivity of the mixture and the sum of the conductivities of the components alone passes through a maximum at a pH theoretically calculable as the isoelectric point of the system. Certain applications of the results are made to the explanation of the behavior of living tissues.

  1. Role of pipecolic acid in the biosynthesis of lysine in Rhodotorula glutinis.

    PubMed

    Kinzel, J J; Bhattacharjee, J K

    1979-05-01

    The role of pipecolic acid in the biosynthesis of lysine was investigated in Rhodotorula glutinis, an aerobic red yeast. Supplementation of pipecolic acid in the minimal medium supported the growth of mutants lys2, lys3, and lys5; alpha-aminoadipic acid supported the growth of lys5; but neither alpha-aminoadipic acid nor pipecolic acid supported the growth of mutants MNNG42 and MNNG37. During the growth of the appropriate mutants, pipecolic acid was removed from the growth medium and the intracellular pool. In tracer experiments, radioactivity from [(14)C]pipecolic acid was selectively incorporated into the cellular lysine of lys5 and the wild-type strain. l-Pipecolic acid-dependent enzyme activity did not require any cofactor and was inhibited by mercuric chloride and potassium cyanide. This activity was present in the wild-type strain and all of the mutants tested and was repressed in mutant lys5 when grown in the presence of higher concentration of lysine. The reaction product of pipecolic acid was converted to saccharopine by lys5 enzyme in the presence of glutamate and reduced nicotin-amide adenine dinucleotide phosphate. Mutant MNNG37 lacked the saccharopine dehydrogenase activity, indicating that this step is involved in the conversion of alpha-aminoadipic acid and pipecolic acid to lysine. Mutants MNNG37 and MNNG42 accumulated a p-dimethylaminobenzaldehyde-reacting product in the culture supernatant and in the intracellular pool. Chromatographic properties of the p-dimethylaminobenzaldehyde adduct and that of the pipecolic acid-dependent reaction product were similar. The reaction product and the accumulation product were characterized on the basis of mass and absorption spectra as alpha-aminoadipic-semialdehyde, which in solution remains in equilibrium with Delta(1)-piperideine-6-carboxylic acid. Since alpha-aminoadipic-semialdehyde is a known intermediate of the alpha-aminoadipic acid pathway for the biosynthesis of lysine, it is concluded that pipecolic

  2. Kinetic and spectral parameters of interaction of Citrobacter freundii methionine γ-lyase with amino acids.

    PubMed

    Morozova, E A; Bazhulina, N P; Anufrieva, N V; Mamaeva, D V; Tkachev, Y V; Streltsov, S A; Timofeev, V P; Faleev, N G; Demidkina, T V

    2010-10-01

    Kinetic parameters of Citrobacter freundii methionine γ-lyase were determined with substrates in γ-elimination reactions as well as the inhibition of the enzyme in the γ-elimination of L-methionine by amino acids with different structure. The data indicate an important contribution of the sulfur atom and methylene groups to the efficiency of binding of substrates and inhibitors. The rate constants of the enzyme-catalyzed exchange of C-α- and C-β-protons with deuterium were determined, as well as the kinetic isotope effect of the deuterium label in the C-α-position of inhibitors on the rate of exchange of their β-protons. Neither stereoselectivity in the β-proton exchange nor noticeable α-isotope effect on the exchange rates of β-protons was found. The ionic and tautomeric composition of the external Schiff base of methionine γ-lyase was determined. Spectral characteristics (absorption and circular dichroism spectra) of complexes with substrates and inhibitors were determined. The spectral and kinetic data indicate that deamination of aminocrotonate should be the rate-determining stage of the enzymatic reaction.

  3. Methionine Biosynthesis in Lemna

    PubMed Central

    Thompson, Gregory A.; Datko, Anne H.; Mudd, S. Harvey; Giovanelli, John

    1982-01-01

    Regulation of enzymes of methionine biosynthesis was investigated by measuring the specific activities of O-phosphohomoserine-dependent cystathionine γ-synthase, O-phosphohomoserine sulfhydrylase, and O-acetylserine sulfhydrylase in Lemna paucicostata Hegelm. 6746 grown under various conditions. For cystathionine γ-synthase, it was observed that (a) adding external methionine (2 μm) decreased specific activity to 15% of control, (b) blocking methionine synthesis with 0.05 μml-aminoethoxyvinylglycine or with 36 μm lysine plus 4 μm threonine (Datko, Mudd 1981 Plant Physiol 69: 1070-1076) caused a 2- to 3-fold increase in specific activity, and (c) blocking methionine synthesis and adding external methionine led to the decreased specific activity characteristic of methionine addition alone. Activity in extracts from control cultures was unaffected by addition of methionine, lysine, threonine, lysine plus threonine, S-adenosylmethionine, or S-methylmethionine sulfonium to the assay mixture. Parallel studies of O-phosphohomoserine sulfhydrylase and O-acetylserine sulfhydrylase showed that O-phosphohomoserine sulfhydrylase activity responded to growth conditions identically to cystathionine γ-synthase activity, whereas O-acetylserine sulfhydrylase activity remained unaffected. Lemna extracts did not catalyze lanthionine formation from O-acetylserine and cysteine. Estimates of kinetic constants for the three enzyme activities indicate that O-acetylserine sulfhydrylase has much higher activity and affinity for sulfide than O-phosphohomoserine sulfhydrylase. The results suggest that (a) methionine, or one of its products, regulates the amount of active cystathionine γ-synthase in Lemna, (b) O-phosphohomoserine sulfhydrylase and cystathionine γ-synthase are probably activities of one enzyme that has low specificity for its sulfur-containing substrate, and (c) O-acetylserine sulfhydrylase is a separate enzyme. The relatively high activity and affinity for sulfide of

  4. Energetics and dynamics of the fragmentation reactions of protonated peptides containing methionine sulfoxide or aspartic acid via energy- and time-resolved surface induced dissociation.

    PubMed

    Lioe, Hadi; Laskin, Julia; Reid, Gavin E; O'Hair, Richard A J

    2007-10-25

    The surface-induced dissociation (SID) of six model peptides containing either methionine sulfoxide or aspartic acid (GAILM(O)GAILR, GAILM(O)GAILK, GAILM(O)GAILA, GAILDGAILR, GAILDGAILK, and GAILDGAILA) have been studied using a specially configured Fourier transform ion-cyclotron resonance mass spectrometer (FT-ICR MS). In particular, we have investigated the energetics and dynamics associated with (i) preferential cleavage of the methionine sulfoxide side chain via the loss of CH3SOH (64 Da), and (ii) preferential cleavage of the amide bond C-terminal to aspartic acid. The role of proton mobility in these selective bond cleavage reactions was examined by changing the C-terminal residue of the peptide from arginine (nonmobile proton conditions) to lysine (partially mobile proton conditions) to alanine (mobile proton conditions). Time- and energy-resolved fragmentation efficiency curves (TFECs) reveal that selective cleavages due to the methionine sulfoxide and aspartic acid residues are characterized by slow fragmentation kinetics. RRKM modeling of the experimental data suggests that the slow kinetics is associated with large negative entropy effects and these may be due to the presence of rearrangements prior to fragmentation. It was found that the Arrhenius pre-exponential factor (A) for peptide fragmentations occurring via selective bond cleavages are 1-2 orders of magnitude lower than nonselective peptide fragmentation reactions, while the dissociation threshold (E0) is relatively invariant. This means that selective bond cleavage is kinetically disfavored compared to nonselective amide bond cleavage. It was also found that the energetics and dynamics for the preferential loss of CH3SOH from peptide ions containing methionine sulfoxide are very similar to selective C-terminal amide bond cleavage at the aspartic acid residue. These results suggest that while preferential cleavage can compete with amide bond cleavage energetically, dynamically, these processes

  5. Energetics and Dynamics of the Fragmentation Reactions of Protonated Peptides Containing Methionine Sulfoxide or Aspartic Acid via Energy- and Time-Resolved Surface Induced Dissociation

    SciTech Connect

    Lioe, Hadi; Laskin, Julia; Reid, Gavin E.; O'Hair, Richard Aj

    2007-10-25

    The surface-induced dissociation (SID) of six model peptides containing either methionine sulfoxide or aspartic acid (GAILM(O)GAILR, GAILM(O)GAILK, GAILM(O)GAILA, GAILDGAILR, GAILDGAILK, and GAILDGAILA) have been studied using a specially configured Fourier transform ion-cyclotron resonance mass spectrometer (FT-ICR MS). In particular, we have investigated the energetics and dynamics associated with (i) preferential cleavage of the methionine sulfoxide side chain via the loss of CH3SOH (64Da), and (ii) preferential cleavage of the amide bond C-terminal to aspartic acid. The role of proton mobility on these selective bond cleavage reactions was examined by changing the C-terminal residue of the peptide from arginine (non-mobile proton conditions) to lysine (partially-mobile proton conditions) to alanine (mobile proton conditions). Time- and energy-resolved fragmentation efficiency curves (TFEC) reveals that selective cleavages due to the methionine sulfoxide and aspartic acid residues are characterized by slow fragmentation kinetics. RRKM modeling of the experimental data suggests that the slow kinetics is associated with large negative entropy effects and these may be due to the presence of rearrangements prior to fragmentation. It was found that the Arrhenius pre-exponential factor (A) for peptide fragmentations occurring via selective bond cleavages are 1–2 orders of magnitude lower than non-selective peptide fragmentation reactions, while the dissociation threshold (E0) is relatively invariant. This means that selective bond cleavage is kinetically disfavored compared to non-selective amide bond cleavage. It was also found that the energetics and dynamics for the preferential loss of CH3SOH from peptide ions containing methionine sulfoxide are very similar to selective C-terminal amide bond cleavage at the aspartic acid residue. These results suggest that while preferential cleavage can compete with amide bond cleavage energetically, dynamically, these

  6. Comparison of methionine sources around requirement levels using a methionine efficacy method in 0 to 28 day old broilers.

    PubMed

    Agostini, P S; Dalibard, P; Mercier, Y; Van der Aar, P; Van der Klis, J D

    2016-03-01

    The addition of methionine in the poultry feed industry is still facing the relative efficacy dilemma between DL-methionine (DLM) and hydroxy-methionine (HMTBA). The aim of this study was to compare the effect of dietary DLM and HMTBA on broiler performance at different levels of total sulfur amino acids (TSAA). The treatments consisted of a basal diet without methionine addition, and 4 increasing methionine doses for both sources resulting in TSAA/Lysine ratios from 0.62 to 0.73 in the starter phase and 0.59 to 0.82 in the grower phase. The comparison of product performance was performed by three-way ANOVA analysis and by methionine efficacy calculation as an alternative method of comparison. Growth results obtained during the starter phase with the different methionine supplementations did not show significant growth responses to TSAA levels, indicating a lower methionine requirement in the starter phase than currently assumed. However, a significant methionine dose effect was obtained for the period 10 to 28 day of age and for the entire growth period of 0 to 28 day of age. Excepting a significant gender effect, the statistical analysis did not allow for the discrimination of methionine sources, and no interaction between source and dose level was observed up to 28 days of age. A significant interaction between source and dose level was observed for methionine efficacy for the grower phase, and the total growth period showed better HMTBA efficacy at higher TSAA value. The exponential model fitted to each methionine source for body weight response depending on methionine intake or for feed conversion ratio (FCR) depending on methionine doses did not allow the methionine sources to be distinguished. Altogether, these results conclude that methionine sources lead to similar performances response when compared at TSAA values around the broiler requirement level. These results also showed that at TSAA values above requirement, HMTBA had a better methionine efficacy

  7. YjeH Is a Novel Exporter of l-Methionine and Branched-Chain Amino Acids in Escherichia coli.

    PubMed

    Liu, Qian; Liang, Yong; Zhang, Yun; Shang, Xiuling; Liu, Shuwen; Wen, Jifu; Wen, Tingyi

    2015-11-01

    Amino acid efflux transport systems have important physiological functions and play vital roles in the fermentative production of amino acids. However, no methionine exporter has yet been identified in Escherichia coli. In this study, we identified a novel amino acid exporter, YjeH, in E. coli. The yjeH overexpression strain exhibited high tolerance to the structural analogues of l-methionine and branched-chain amino acids, decreased intracellular amino acid levels, and enhanced export rates in the presence of a Met-Met, Leu-Leu, Ile-Ile, or Val-Val dipeptide, suggesting that YjeH functions as an exporter of l-methionine and the three branched-chain amino acids. The export of the four amino acids in the yjeH overexpression strain was competitively inhibited in relation to each other. The expression of yjeH was strongly induced by increasing cytoplasmic concentrations of substrate amino acids. Green fluorescent protein (GFP)-tagged YjeH was visualized by total internal reflection fluorescence microscopy to confirm the plasma membrane localization of YjeH. Phylogenetic analysis of transporters indicated that YjeH belongs to the amino acid efflux family of the amino acid/polyamine/organocation (APC) superfamily. Structural modeling revealed that YjeH has the typical "5 + 5" transmembrane α-helical segment (TMS) inverted-repeat fold of APC superfamily transporters, and its binding sites are strictly conserved. The enhanced capacity of l-methionine export by the overexpression of yjeH in an l-methionine-producing strain resulted in a 70% improvement in titer. This study supplements the transporter classification and provides a substantial basis for the application of the methionine exporter in metabolic engineering.

  8. Effect of increased methionine level on performance and apparent ileal digestibility of amino acids in ducks.

    PubMed

    Jamroz, D; Wiliczkiewicz, A; Lemme, A; Orda, J; Skorupińska, J; Wertelecki, T

    2009-10-01

    The experiment was conducted with 960 one-day-old ducklings fed mixtures (I control - 0.28% methionine) additionally supplemented with DL-methionine (DL-Met) at amounts: 0.03% (group II), 0.07% (III), 0.12% (IV) and 0.18% (V). The performance, carcass quality and apparent ileal digestibility of amino acids as the criterions of methionine (Met) effectivity were considered. The analysis of growth and development of ducks as an effect of diversified DL-Met supplements indicate that increased content of this amino acid in the diets has not affected clearly the performance parameters. The body weight of 21-day-old ducklings was significantly affected only by the level of 0.12% of added Met in comparison to control group. On day 42, the differences among groups were negligible; only the addition of 0.12% DL-Met has increased the body weight by 2.4% when compared with control (p > 0.05). Feed conversion estimated for a period of 1-42 days has not been influenced by Met supplementation. The indistinct, however, visible tendency of better ileal amino acids' apparent digestibility (for Asp.a.,Thr, Ser, Glu, Lys) was noted in the groups fed supplemented diets. Application of 0.07% and 0.18% of DL-met, has significantly (p < 0.05) improved the coefficient of cysteine (Cys) apparent ileal digestibility; however, the improvement of Met apparent ileal digestibility has been achieved by the addition of 0.18% Met. The mortality of ducklings in the experiment was very low and varied between 3.15% (II) and 0.0% (groups I and III). In general, application of 0.12% of DL-Met to mixture containing 0.28% Met had positive effect on the productive output of birds and also improved the apparent ileal digestibility of Cys and Met.

  9. Self-assembly and foaming properties of fatty acid-lysine aqueous dispersions.

    PubMed

    Novales, Bruno; Riaublanc, Alain; Navailles, Laurence; Houssou, Bérénice Houinsou; Gaillard, Cédric; Nallet, Frédéric; Douliez, Jean-Paul

    2010-04-20

    We report on dispersions of fatty acid-lysine salts in aqueous solutions which are further used to produce foams. The alkyl chain length is varied from dodecyl to stearic. In aqueous solutions, the lysine salt of the dodecyl chain yields an isotropic solution, probably micelles, whereas for longer alkyl chains, vesicles formed but crystallized upon resting at room temperature or when kept at 4 degrees C. Solid-state NMR showed that in vesicles fatty acids are embedded in a lamellar arrangement passing from a gel to a fluid state upon heating; the transition temperature at which it occurs was determined by DSC. Those results are confirmed by small-angle neutron scattering which also give additional information on the bilayer structure. Incredibly stable foams are obtained using the palmitic acid/Lys salt whereas for other alkyl chain length, poor or no foam is formed. We conclude that the foamability is related to the phase behavior in aqueous solution. PMID:20334439

  10. Amino acid metabolism in the human fetus at term: leucine, valine, and methionine kinetics.

    PubMed

    van den Akker, Chris H P; Schierbeek, Henk; Minderman, Gardi; Vermes, Andras; Schoonderwaldt, Ernst M; Duvekot, Johannes J; Steegers, Eric A P; van Goudoever, Johannes B

    2011-12-01

    Human fetal metabolism is largely unexplored. Understanding how a healthy fetus achieves its fast growth rates could eventually play a pivotal role in improving future nutritional strategies for premature infants. To quantify specific fetal amino acid kinetics, eight healthy pregnant women received before elective cesarean section at term, continuous stable isotope infusions of the essential amino acids [1-13C,15N]leucine, [U-13C5]valine, and [1-13C]methionine. Umbilical blood was collected after birth and analyzed for enrichments and concentrations using mass spectrometry techniques. Fetuses showed considerable leucine, valine, and methionine uptake and high turnover rates. α-Ketoisocaproate, but not α-ketoisovalerate (the leucine and valine ketoacids, respectively), was transported at net rate from the fetus to the placenta. Especially, leucine and valine data suggested high oxidation rates, up to half of net uptake. This was supported by relatively low α-ketoisocaproate reamination rates to leucine. Our data suggest high protein breakdown and synthesis rates, comparable with, or even slightly higher than in premature infants. The relatively large uptakes of total leucine and valine carbon also suggest high fetal oxidation rates of these essential branched chain amino acids.

  11. Effects of calcium soaps of rapeseed fatty acids and protected methionine on milk yield and composition in dairy cows.

    PubMed

    Kowalski, Z M; Pisulewski, P M; Spanghero, M

    1999-11-01

    The objective of this study was to determine the effects of supplementing the diets of dairy cows with Ca soaps of rapeseed fatty acids (CSRFA) and rumen-protected (RP) methionine on their milk yield and composition, including milk protein fractions and fatty acids. Twelve Polish Red Lowland cows were used in a complete balanced two period changeover experiment. The four treatment diets were a control consisting of a total mixed ration of grass silage and concentrates, and the total mixed ration supplemented with RP methionine, CSRFA or RP methionine plus CSRFA. Dry matter intake was not affected by diet. Milk yield increased when cows were given the diet with CSRFA, but supplementation of diets with RP methionine did not affect milk yield. Milk protein content, but not milk protein yield, decreased when CSRFA was given. The addition of RP methionine to the control diet and the CSRFA diet produced similar increases in the milk protein content. Supplementation of the diet with CSRFA significantly changed the milk fatty acid profile: the proportions of 10:0, 12:0, 14:0, 15:0 and 16:0 in milk fat decreased, but those of 18:0 and cis-18:1 increased. We conclude that CSRFA can be used in practical dairy diets to increase milk yield and manipulate its fatty acid composition.

  12. In vitro degradation of lysine by ruminal fluid-based fermentations and by Fusobacterium necrophorum.

    PubMed

    Elwakeel, E A; Amachawadi, R G; Nour, A M; Nasser, M E A; Nagaraja, T G; Titgemeyer, E C

    2013-01-01

    The objective of these studies was to characterize some factors affecting lysine degradation by mixed ruminal bacteria and by ruminal Fusobacterium necrophorum. Mixed ruminal bacteria degraded lysine, and addition of pure cultures of F. necrophorum did not increase lysine degradation. Addition of acetic or propionic acid strikingly reduced NH(3) production from lysine by mixed ruminal bacteria at pH 6, but not at pH 7. Although typical ruminal environments with acidic pH and normal concentrations of volatile fatty acids might inhibit lysine degradation by F. necrophorum, ruminal fluid contained enough bacteria with a lysine-degrading capacity to ferment 50 mM lysine in vitro. Of 7 strains of ruminal F. necrophorum tested, all grew on both lactate and lysine as the primary energy source. Both subspecies of ruminal F. necrophorum (necrophorum and funduliforme) used lysine as a primary C and energy source. Lysine and glutamic acid were effectively fermented by F. necrophorum, but alanine and tryptophan were not, and histidine and methionine were fermented only to a minor extent. The end products of lactate fermentation by F. necrophorum were propionate and acetate, and those of lysine degradation were butyrate and acetate. Fermentation of glutamic acid by F. necrophorum yielded acetate and butyrate in a ratio near to 2:1. The minimum inhibitory concentration of tylosin for F. necrophorum was not dependent on whether bacteria were grown with lactate or lysine, but F. necrophorum was more susceptible to monensin when grown on lysine than on lactate. Although F. necrophorum is generally resistant to monensin, the ionophore may reduce lysine degradation by F. necrophorum in the rumen. The essential oil components limonene, at 20 or 100 μg/mL, and thymol, at 100 μg/mL, inhibited F. necrophorum growth, whereas eugenol, guaiacol, and vanillin had no effect. Our findings may lead to ways to minimize ruminal lysine degradation and thus increase its availability to the animal

  13. Controlled trial of whole body protein synthesis and plasma amino acid concentrations in yearling horses fed graded amounts of lysine.

    PubMed

    Mastellar, S L; Coleman, R J; Urschel, K L

    2016-10-01

    Lysine has been reported as the first limiting amino acid in typical equine diets. Indicator amino acid oxidation (IAAO) has become the standard method for determining amino acid requirements in other species, but prior to this study, it has not been used to determine equine requirements. The aim of this study was to evaluate whole body protein synthesis and plasma and muscle amino acid concentrations in response to graded levels of lysine intake in yearling horses. Six Thoroughbred colts (358 ± 5 kg) were fed each of six treatment lysine intakes ranging from 76 to 136 mg/kg body weight/day. Blood samples were taken before and 90 min after the morning concentrate meal. Gluteal muscle biopsies were taken ~100 min after the morning concentrate meal. The next day, whole body phenylalanine kinetics were determined using a 2 h primed, constant infusion of [(13)C] sodium bicarbonate followed by a 6 h primed, constant infusion of [1-(13)C] phenylalanine. Plasma lysine concentrations increased linearly (P <0.05) at both the 0 and 90 min time points with increasing lysine intakes. Free muscle asparagine, aspartate, arginine, glutamine, lysine, taurine and tryptophan concentrations responded quadratically to lysine intake (P <0.05). Phenylalanine kinetics did not differ between treatment intakes (P > 0.10). A broken line analysis of lysine intake and phenylalanine oxidation failed to yield a breakpoint from which to determine a lysine requirement. These diets may have been limiting in an amino acid other than lysine, underscoring the lack of data concerning amino acid requirements and bioavailability data in the horse. PMID:27687933

  14. Controlled trial of whole body protein synthesis and plasma amino acid concentrations in yearling horses fed graded amounts of lysine.

    PubMed

    Mastellar, S L; Coleman, R J; Urschel, K L

    2016-10-01

    Lysine has been reported as the first limiting amino acid in typical equine diets. Indicator amino acid oxidation (IAAO) has become the standard method for determining amino acid requirements in other species, but prior to this study, it has not been used to determine equine requirements. The aim of this study was to evaluate whole body protein synthesis and plasma and muscle amino acid concentrations in response to graded levels of lysine intake in yearling horses. Six Thoroughbred colts (358 ± 5 kg) were fed each of six treatment lysine intakes ranging from 76 to 136 mg/kg body weight/day. Blood samples were taken before and 90 min after the morning concentrate meal. Gluteal muscle biopsies were taken ~100 min after the morning concentrate meal. The next day, whole body phenylalanine kinetics were determined using a 2 h primed, constant infusion of [(13)C] sodium bicarbonate followed by a 6 h primed, constant infusion of [1-(13)C] phenylalanine. Plasma lysine concentrations increased linearly (P <0.05) at both the 0 and 90 min time points with increasing lysine intakes. Free muscle asparagine, aspartate, arginine, glutamine, lysine, taurine and tryptophan concentrations responded quadratically to lysine intake (P <0.05). Phenylalanine kinetics did not differ between treatment intakes (P > 0.10). A broken line analysis of lysine intake and phenylalanine oxidation failed to yield a breakpoint from which to determine a lysine requirement. These diets may have been limiting in an amino acid other than lysine, underscoring the lack of data concerning amino acid requirements and bioavailability data in the horse.

  15. Linkage between the bacterial acid stress and stringent responses: the structure of the inducible lysine decarboxylase.

    PubMed

    Kanjee, Usheer; Gutsche, Irina; Alexopoulos, Eftichia; Zhao, Boyu; El Bakkouri, Majida; Thibault, Guillaume; Liu, Kaiyin; Ramachandran, Shaliny; Snider, Jamie; Pai, Emil F; Houry, Walid A

    2011-03-01

    The Escherichia coli inducible lysine decarboxylase, LdcI/CadA, together with the inner-membrane lysine-cadaverine antiporter, CadB, provide cells with protection against mild acidic conditions (pH∼5). To gain a better understanding of the molecular processes underlying the acid stress response, the X-ray crystal structure of LdcI was determined. The structure revealed that the protein is an oligomer of five dimers that associate to form a decamer. Surprisingly, LdcI was found to co-crystallize with the stringent response effector molecule ppGpp, also known as the alarmone, with 10 ppGpp molecules in the decamer. ppGpp is known to mediate the stringent response, which occurs in response to nutrient deprivation. The alarmone strongly inhibited LdcI enzymatic activity. This inhibition is important for modulating the consumption of lysine in cells during acid stress under nutrient limiting conditions. Hence, our data provide direct evidence for a link between the bacterial acid stress and stringent responses. PMID:21278708

  16. Enzymatic preparation of. cap alpha. - and. beta. -deuterated or tritiated amino acids with l-methionine. gamma. -lyase

    SciTech Connect

    Esaki, N.; Sawada, S.; Tanaka, H.; Soda, K.

    1982-01-15

    L-Methionine ..gamma..-lyase catalyzes the exchange of ..cap alpha..- and ..beta..-hydrogens of L-methionine and S-methyl-L-cysteine with deuterium or tritium of solvents. The rate of ..cap alpha..-hydrogen exchange with deuterium was about 40 times faster than that of the elimination reactions. The deuterium and tritium were exchanged also with the ..cap alpha..- and ..beta..-hydrogens of the straight-chain amino acids which do not undergo the elimination: L-alanine, L-..cap alpha..-aminobutyrate, L-norvaline, and L-norleucine. No exchange occurs for the D-isomers, acidic L-amino acids, basic L-amino acids, and branched-chain L-amino acids, although ..cap alpha..-hydrogen of glycine, L-trypotophan, and L-phenylalanine is exchanged slowly. These enzymatic hydrogen-exchange reactions facilitate specific labeling of the L-amino acids with deuterium and tritium.

  17. Amino acid metabolism in the piglet. 3. Influence of lysine level in the diet on energy metabolism and in vivo oxidation.

    PubMed

    Chavez, E R; Bayley, H S

    1976-11-01

    1. Supplementing a lysine-deficient diet (5 g lysine/kg) with five increments of lysine, each of 2 g/kg, resulted in increased in growth rate of Yorkshire piglets, aged between 3 and 7 weeks, up to the highest level of lysine (15 g/kg). 2. The free lysine concentration of plasma tended to increase as the dietary lysine level increased from 13 to 15 g/kg, and plasma threonine concentration decreased significantly as the lysine content of the diet was increased from 11 to 15 g/kg indicating that threonine was the second limiting amino acid in the diet. 3. Oxygen consumption and carbon dioxide production of the piglets were not influenced by supplementing the diets with lysine. The heat production was 0-313 kJ/min per kg body-weight in the 6 h experimental period. 4. Supplementation of the diet with lysine had no consistent effect on the recovery of 14C as 14CO2 from a single dose of L-[U-14C]lysine. 5. Adjustment of the determined recoveries of the tracer dose of lysine for the differences in the plasma concentrations of free lysine for the pigs receiving the graded levels of dietary lysine simplified the relationship between recovery and dietary lysine level: it was linear for the first four increments in dietary lysine and then increased sharply for the fifth increment. This indicated that a marked change in the rate of lysine catabolism occurred as the level of dietary lysine was increased from 13 to 15 g/kg. 6. The results of this experiment indicate that the piglets' requirement for lysine is between 13 and 15 g lysine/kg in a diet which contained 181 g crude protein (nitrogen X6-25)/kg.

  18. A Single Amino Acid Change Is Responsible for Evolution of Acyltransferase Specificity in Bacterial Methionine Biosynthesis

    SciTech Connect

    Zubieta, C.; Arkus, K.A.J.; Cahoon, R.E.; Jez, J.M.

    2009-05-28

    Bacteria and yeast rely on either homoserine transsuccinylase (HTS, metA) or homoserine transacetylase (HTA; met2) for the biosynthesis of methionine. Although HTS and HTA catalyze similar chemical reactions, these proteins are typically unrelated in both sequence and three-dimensional structure. Here we present the 2.0 {angstrom} resolution x-ray crystal structure of the Bacillus cereus metA protein in complex with homoserine, which provides the first view of a ligand bound to either HTA or HTS. Surprisingly, functional analysis of the B. cereus metA protein shows that it does not use succinyl-CoA as a substrate. Instead, the protein catalyzes the transacetylation of homoserine using acetyl-CoA. Therefore, the B. cereus metA protein functions as an HTA despite greater than 50% sequence identity with bona fide HTS proteins. This result emphasizes the need for functional confirmation of annotations of enzyme function based on either sequence or structural comparisons. Kinetic analysis of site-directed mutants reveals that the B. cereus metA protein and the E. coli HTS share a common catalytic mechanism. Structural and functional examination of the B. cereus metA protein reveals that a single amino acid in the active site determines acetyl-CoA (Glu-111) versus succinyl-CoA (Gly-111) specificity in the metA-like of acyltransferases. Switching of this residue provides a mechanism for evolving substrate specificity in bacterial methionine biosynthesis. Within this enzyme family, HTS and HTA activity likely arises from divergent evolution in a common structural scaffold with conserved catalytic machinery and homoserine binding sites.

  19. Effects of Supplemental Liquid DL-methionine Hydroxy Analog Free Acid in Diet on Growth Performance and Gastrointestinal Functions of Piglets

    PubMed Central

    Kaewtapee, C.; Krutthai, N.; Bunchasak, C.

    2016-01-01

    This study was conducted to determine the effect of dietary supplementation of liquid DL-methionine hydroxy analog free acid (DL-MHA) on growth performance and gastrointestinal conditions of piglets. One hundred and eighty crossbred barrow piglets (Large White×Landrace, body weight: 12.48±0.33 kg) were divided into three groups with ten replications of six piglets each. Piglets received DL-MHA in diet at a concentration of 0 (control group), 0.15%, or 0.24%. The results indicated that increasing the standardized ileal digestible (SID) of sulfur amino acids (SAA) to lysine (SID SAA:Lys) ratio by supplementation of DL-MHA tended to increase (quadratic; p<0.10) weight gain and ADG, and showed slightly greater (linear; p<0.10) gain:feed ratio. The pH in the diet and cecum linearly decreased (p<0.01), whereas pH in colon had a quadratic response (p<0.01) with increasing supplementation of DL-MHA. By greater supplementation of DL-MHA, the population of Lactobacillus spp. in rectum was likely to increase (quadratic; p<0.10), but Escherichia coli population in the diet was reduced (quadratic; p<0.05). Acetic acid concentration and total short-chain fatty acids in cecum linearly increased (p<0.05), whereas valeric acid in cecum quadratically increased (p<0.05) with increasing DL-MHA levels. Moreover, the villous height of the jejunum quadratically increased (p<0.01) as the supplementation of DL-MHA was increased. It is concluded that the addition of DL-MHA in diet improved the growth performance and the morphology of gastrointestinal tract of piglets. PMID:26954213

  20. Chiral effects in amino acid adsorption on Au(111): A comparison of cysteine, homocysteine and methionine

    NASA Astrophysics Data System (ADS)

    Popa, Tatiana; Ting, Elvis C. M.; Paci, Irina

    2014-11-01

    A combined classical/quantum methodology is used to examine chiral effects upon adsorption of three sulfur-containing amino acids on the Au(111) surface: cysteine, homocysteine and methionine. Parallel tempering Monte Carlo simulations were employed to broadly examine the configurational space of monomers, dimers and trimers of the molecules on the gold surface. Density functional theory was applied to promising structural targets in order to incorporate higher order electronic structure effects in a study of relative stabilities of the various molecular states upon adsorption. As the precursors of chiral structure formation, like and unlike dimers were investigated at some length, with consideration given to the mode of sorption (chemisorption of physisorption) and the existence of zwitterionic states. We found that neutral (non-zwitterionic) molecules adsorbed weakly on the highly-coordinated Au(111) surfaces. As a consequence, pair configurations in dimers were insufficiently constrained to lead to differential stabilities of homochiral and heterochiral dimers. Whereas neutral molecule interactions were non-discriminating, strong chiral discrimination was found in zwitterionic amino acids. The zwitterionic forms of the larger molecules equilibrated closer to the surface, and the stronger molecule-molecule and molecule-surface interactions were such that homochiral dimers were stable whereas heterochiral dimers were not.

  1. Sulfur amino acids and atherosclerosis: a role for excess dietary methionine.

    PubMed

    Selhub, Jacob; Troen, Aron M

    2016-01-01

    The homocysteine theory of arteriosclerosis received credence when it was shown that after a methionine load, circulating homocysteine-cysteine concentrations were higher in cardiovascular disease patients than in healthy controls. Subsequent studies showing associations between homocysteine and coronary artery disease, stroke and cognitive impairment, relied on small increases in homocysteine concentration unlike the very high homocysteine seen in the rare genetic disorders that lead to homocystinuria and much higher homocysteine levels. Subsequent studies in cell culture, animals, and humans showed that a variety of cardiovascular adverse effects of "high homocysteine" introduced either as a nonphysiological bolus or as a methionine load led to high homocysteine. We fed apolipoprotein E-deficient mice diets designed to achieve three conditions: (1) high methionine intake with normal blood homocysteine, (2) high methionine intake with B vitamin deficiency and hyperhomocysteinemia, and (3) normal methionine intake with both B vitamin deficiency and hyperhomocysteinemia. We found that the mice fed methionine-rich diets had significant atheromatous pathology in the aortic arch even with normal plasma homocysteine levels. Mice fed B vitamin-deficient diets developed severe hyperhomocysteinemia but without any increase in vascular pathology. Our findings suggest that even moderate increases in methionine intake are atherogenic in susceptible mice while high plasma homocysteine is not.

  2. Engineering Corynebacterium glutamicum for fast production of L-lysine and L-pipecolic acid.

    PubMed

    Pérez-García, Fernando; Peters-Wendisch, Petra; Wendisch, Volker F

    2016-09-01

    The Gram-positive Corynebacterium glutamicum is widely used for fermentative production of amino acids. The world production of L-lysine has surpassed 2 million tons per year. Glucose uptake and phosphorylation by C. glutamicum mainly occur by the phosphotransferase system (PTS) and to lesser extent by inositol permeases and glucokinases. Heterologous expression of the genes for the high-affinity glucose permease from Streptomyces coelicolor and Bacillus subtilis glucokinase fully compensated for the absence of the PTS in Δhpr strains. Growth of PTS-positive strains with glucose was accelerated when the endogenous inositol permease IolT2 and glucokinase from B. subtilis were overproduced with balanced translation initiation rates using plasmid pEKEx3-IolTBest. When the genome-reduced C. glutamicum strain GRLys1 carrying additional in-frame deletions of sugR and ldhA to derepress glycolytic and PTS genes and to circumvent formation of L-lactate as by-product was transformed with this plasmid or with pVWEx1-IolTBest, 18 to 20 % higher volumetric productivities and 70 to 72 % higher specific productivities as compared to the parental strain resulted. The non-proteinogenic amino acid L-pipecolic acid (L-PA), a precursor of immunosuppressants, peptide antibiotics, or piperidine alkaloids, can be derived from L-lysine. To enable production of L-PA by the constructed L-lysine-producing strain, the L-lysine 6-dehydrogenase gene lysDH from Silicibacter pomeroyi and the endogenous pyrroline 5-carboxylate reductase gene proC were overexpressed as synthetic operon. This enabled C. glutamicum to produce L-PA with a yield of 0.09 ± 0.01 g g(-1) and a volumetric productivity of 0.04 ± 0.01 g L(-1) h(-1).To the best of our knowledge, this is the first fermentative process for the production of L-PA from glucose.

  3. Photooxidation of Methionine

    ERIC Educational Resources Information Center

    Lewis, Catherine; Scouten, William H.

    1976-01-01

    Describes an experiment in which the photooxidation of methionine using free methylene blue as the sensitizer is applied to the isolated amino acid or to the methionyl residues of a complex polypeptide. (MLH)

  4. Synthesis of peptides from amino acids and ATP with lysine-rich proteinoid

    NASA Technical Reports Server (NTRS)

    Nakashima, T.; Fox, S. W.

    1980-01-01

    The paper examines the synthesis of peptides from aminoacids and ATP with a lysine-rich protenoid. The latter in aqueous solution catalyzes the formation of peptides from free amino acids and ATP; this catalytic activity is not found in acidic protenoids, even though the latter contain a basic aminoacid. The pH optimum for the synthesis is about 11, but it is appreciable below 8 and above 13. Temperature data indicate an optimum at 20 C or above, with little increase in rate up to 60 C. Pyrophosphate can be used instead of ATP, but the yields are lower. The ATP-aided syntheses of peptides in aqueous solution occur with several types of proteinous aminoacids.

  5. Evaluation of protein content, lysine and sulfur-containing amino acids content and electrophoretic patterns of soluble proteins for gamma-irradiated semolina before and after milling of durum wheat

    NASA Astrophysics Data System (ADS)

    Azzeh, F. S.; Amr, A. S.

    2009-11-01

    Influenced of gamma irradiation (0, 0.25, 1, 2.5, 5 and 10 kGy) on total nitrogen, lysine and sulfur-containing amino acids content and electrophoretic patterns of soluble proteins of semolina was studied. The effect of irradiation before and after milling on previous parameters was also investigated. Protein content of semolina was not affected with gamma irradiation before and after milling. Up to 10 kGy dose, cystine and methionine were not significantly changed, although they increased slightly with increasing irradiation dose. Lysine content decreased significantly ( P≤0.05) at irradiation dose higher than 5 kGy. At 10 kGy dose, lysine decreased 5% and 14% for irradiated semolina and that obtained from irradiated wheat grains, respectively. The bands number and intensity of soluble proteins decreased with increasing irradiation dose higher than 5 kGy, as shown on SDS-PAGE electrophoresis. Irradiated semolina and semolina obtained from irradiated wheat grains at 10 kGy showed 13 and 15 bands, respectively. Unirradiated sample showed 19 bands.

  6. Herbivore-Induced SABATH Methyltransferases of Maize That Methylate Anthranilic Acid Using S-Adenosyl-l-Methionine1[W

    PubMed Central

    Köllner, Tobias G.; Lenk, Claudia; Zhao, Nan; Seidl-Adams, Irmgard; Gershenzon, Jonathan; Chen, Feng; Degenhardt, Jörg

    2010-01-01

    Volatile methyl esters are common constituents of plant volatiles with important functions in plant defense. To study the biosynthesis of these compounds, especially methyl anthranilate and methyl salicylate, we identified a group of methyltransferases that are members of the SABATH enzyme family in maize (Zea mays). In vitro biochemical characterization after bacterial expression revealed three S-adenosyl-l-methionine-dependent methyltransferases with high specificity for anthranilic acid as a substrate. Of these three proteins, Anthranilic Acid Methyltransferase1 (AAMT1) appears to be responsible for most of the S-adenosyl-l-methionine-dependent methyltransferase activity and methyl anthranilate formation observed in maize after herbivore damage. The enzymes may also be involved in the formation of low amounts of methyl salicylate, which are emitted from herbivore-damaged maize. Homology-based structural modeling combined with site-directed mutagenesis identified two amino acid residues, designated tyrosine-246 and glutamine-167 in AAMT1, which are responsible for the high specificity of AAMTs toward anthranilic acid. These residues are conserved in each of the three main clades of the SABATH family, indicating that the carboxyl methyltransferases are functionally separated by these clades. In maize, this gene family has diversified especially toward benzenoid carboxyl methyltransferases that accept anthranilic acid and benzoic acid. PMID:20519632

  7. Effects of dietary lysine levels on plasma free amino acid profile in late-stage finishing pigs.

    PubMed

    Regmi, Naresh; Wang, Taiji; Crenshaw, Mark A; Rude, Brian J; Wu, Guoyao; Liao, Shengfa F

    2016-01-01

    Muscle growth requires a constant supply of amino acids (AAs) from the blood. Therefore, plasma AA profile is a critical factor for maximizing the growth performance of animals, including pigs. This research was conducted to study how dietary lysine intake affects plasma AA profile in pigs at the late production stage. Eighteen crossbred (Large White × Landrace) finishing pigs (nine barrows and nine gilts; initial BW 92.3 ± 6.9 kg) were individually penned in an environment controlled barn. Pigs were assigned randomly to one of the three dietary treatments according to a randomized complete block design with sex as block and pig as experiment unit (6 pigs/treatment). Three corn- and soybean meal-based diets contained 0.43 % (lysine-deficient, Diet I), 0.71 % (lysine-adequate, Diet II), and 0.98 % (lysine-excess, Diet III) l-lysine, respectively. After a 4-week period of feeding, jugular vein blood samples were collected from the pigs and plasma was obtained for AA analysis using established HPLC methods. The change of plasma lysine concentration followed the same pattern as that of dietary lysine supply. The plasma concentrations of threonine, histidine, phenylalanine, isoleucine, valine, arginine, and citrulline of pigs fed Diet II or III were lower (P < 0.05) than that of pigs fed Diet I. The plasma concentrations of alanine, glutamate, and glycine of pigs fed Diet II or III were higher (P < 0.05) than that of pigs fed Diet I. The change of plasma leucine and asparagine concentrations followed the patterns similar to that of plasma lysine. Among those affected AAs, arginine was decreased (P < 0.05) in the greatest proportion with the lysine-excess diet. We suggest that the skeletal muscle growth of finishing pigs may be further increased with a lysine-excess diet if the plasma concentration of arginine can be increased through dietary supplementation or other practical nutritional management strategies. PMID:27386336

  8. Methionine, folic acid and vitamin B12 in growing-finishing pigs: impact on growth performance and meat quality.

    PubMed

    Giguére, Alain; Girard, Christiane L; Matte, J Jacques

    2008-06-01

    Growth performance, metabolic variables, and meat quality were measured in 78 growing-finishing pigs using supplements of 0 (C), or 0.2% of DL-methionine (M), and three combinations of folic acid [mg/kg] and cyanocobalamin [microg/kg], respectively 0 and 0 (V0), 10 and 25 (V1), and 10 and 150 (V2) in a 2 x 3 factorial arrangement. Feed conversion was lower (p = 0.05) in M than in C pigs during the growing period (0-4 weeks). Both V1 and V2 treatments increased plasma vitamin B12 (p < 0.01) and decreased plasma homocysteine (p < 0.01). Plasma 5-methyl-tetrahydrofolates were the lowest, highest and intermediate in V0, V1 and V2 pigs (p < 0.04), respectively. In V2 meat, folates were 32% higher, vitamin B12, 55% higher and homocysteine, 28% lower than in V0 (p < 0.01). Oxidative stability of the fresh meat was similar among treatments during a storage period of 42 days. Therefore, methionine supplements improved growth performance during the growing period. Vitamin supplements interacted with the methionine cycle pathway, increased vitamin content of pork meat but did not improve oxidative stability of the fresh meat during storage.

  9. The role of amino acid residues in the active site of L-methionine γ-lyase from Pseudomonas putida.

    PubMed

    Fukumoto, Mitsuki; Kudou, Daizou; Murano, Shouko; Shiba, Tomoo; Sato, Dan; Tamura, Takashi; Harada, Shigeharu; Inagaki, Kenji

    2012-01-01

    Cys116, Lys240*, and Asp241* (asterisks indicate residues from the second subunit of the active dimer) at the active site of L-methionine γ-lyase of Pseudomonas putida (MGL_Pp) are highly conserved among heterologous MGLs. In a previous study, we found that substitution of Cys116 for His led to a drastic increase in activity toward L-cysteine and a decrease in that toward L-methionine. In this study, we examined some properties of the C116H mutant by kinetic analysis and 3D structural analysis. We assumed that substitution of Cys116 for His broke the original hydrogen-bond network and that this induced a significant effect of Tyr114 as a general acid catalyst, possibly due to the narrow space in the active site. The C116H mutant acquired a novel β-elimination activity and lead a drastic conformation change in the histidine residue at position 116 by binding the substrate, suggesting that this His residue affects the reaction specificity of C116H. Furthermore, we suggest that Lys240* is important for substrate recognition and structural stability and that Asp241* is also involved in substrate specificity in the elimination reaction. Based on this, we suggest that the hydrogen-bond network among Cys116, Lys240*, and Asp241* contributes to substrate specificity that is, to L-methionine recognition at the active site in MGL_Pp.

  10. Apparent absorption of methionine and 2-hydroxy-4-methylthiobutanoic acid from gastrointestinal tract of conventional and gnotobiotic pigs.

    PubMed

    Malik, G; Hoehler, D; Rademacher, M; Drew, M D; Van Kessel, A G

    2009-10-01

    The effect of commensal microbiota and feeding corn or wheat/barley-based diets on the apparent gastrointestinal absorption of dl-methionine (MET) and 2-hydroxy-4-methylthiobutanoic acid (MHA-FA) was studied in conventional (n = 32) and gnotobiotic pigs (n = 24). Conventional pigs (CON) were vaginally delivered and sow-reared until weaning at 14 days of age. Gnotobiotic pigs were derived by caesarian section and reared in HEPA (high efficiency particulate air)-filtered isolator units with ad libitum access to a milk-based formula. Corn or wheat/barley-based diets were fed to all pigs from 14 to 24 days of age. At 24 days of age, after an overnight fast, pigs were fed 20 g/kg BW of experimental diet supplemented with 107 Bq of either 3H-l-MET or 3H-l-MHA-FA per kg of feed and chromic oxide (0.5% wt/wt). Pigs were killed for sample collection 3 h after consuming the meal. Residual 3H-MET and 3H-MHA-FA were estimated in gastrointestinal contents as the ratio of 3H : chromic oxide in digesta samples to the ratio of 3H : chromic oxide in feed. In CON pigs, feeding a wheat/barley-based diet increased (P < 0.05) total aerobes, whereas supplementation with MHA-FA increased (P < 0.05) total aerobes and lactobacilli populations in proximal small intestine (SI). Among the gnotobiotic pigs, bacterial contamination occurred such that eight pigs (two isolators) were monoassociated with a Gram-negative bacteria closely related to Providencia spp. and 16 pigs (four isolators) were monoassociated with Gram positive Enterococcus faecium. Species of monoassociated bacterial contaminant and diet composition did not affect residual methionine or MHA-FA in digesta. In both CON and monoassociated (MA) pigs, methionine and MHA-FA were retained in stomach (92%) but disappeared rapidly from proximal SI. Residual methionine and MHA-FA in digesta was not different in MA pigs; however, in CON pigs, less (P < 0.01) apparent residual methionine was found in digesta recovered at 25% (from cranial

  11. Effect on days of lactation and methionine hydroxy analog on incorporation of plasma fatty acids into plasma triglycerides

    SciTech Connect

    Pullen, D.L.; Emergy, R.S. ); Palmquist, D.L. )

    1989-01-01

    Methionine hydroxy analog has been proposed to stimulate hepatic lipoprotein synthesis and incorporation of plasma fatty acids into plasma triglyceride. Seven cows were fed diets containing 0 to 30 g analog/d starting 14 d prepartum. At approximately 30 and 60 d postpartum, cows were continuously infused intravenously with 1-({sup 14}C)palmitic acid for 160 min to achieve steady-state labeling of plasma fatty acid and triglyceride. Turnover of fatty acid and transfer quotients for triglyceride and CO{sub 2} were 3.3 an 2.7 mmol min{sup {minus}1}; 13.0 and 10.0%; and 8.0 and 5.0%, for control and analog, respectively. Proportion of fatty acid turnover incorporated into triglyceride and CO{sub 2} were 14.0 and 15.0%; and 21.0 and 18.0, respectively, for control and analog. Analog increased {sup 14}C recovered in milk fat (52 vs. 36%). Plasma concentration of fatty acids, percent oxidized to CO{sub 2}, and percent of CO{sub 2} from fatty acids decreased with increasing lactation days. Milk fat percent and yield fatty acid turnover, and oxidation were positively correlated with concentration of plasma fatty acids, whereas fatty acid incorporated into plasma triglyceride was negatively correlated with fatty acid concentration. The data suggest that hepatic triglyceride secretion is not increased in early lactation; further, no effects of analog on lipid metabolism were detected.

  12. Identification and Quantitation of the Lipation Product 2-Amino-6-(3-methylpyridin-1-ium-1-yl)hexanoic Acid (MP-Lysine) in Peanuts.

    PubMed

    Globisch, Martin; Deuber, Meike; Henle, Thomas

    2016-08-31

    The lipid peroxidation product acrolein was semiquantitated by GC-MS (EI) in unheated and heated peanut oil, respectively, representing a model system for peanut roasting. Depending on the heating time, acrolein levels significantly increased from 0.2 to 10.7 mg/kg oil. As a result of heating N(α)-acetyl-l-lysine and acrolein, the pyridinium derivative 2-acetamido-6-(3-methylpyridin-1-ium-1-yl)hexanoic acid (MP-acetyl lysine) was identified. In addition, the lysine derivative 2-amino-6-[5-(hydroxymethyl)-3,6-dihydro-2H-pyridin-1-yl]hexanoic acid was identified after reduction and hydrolysis. After preparation of 2-amino-6-(3-methylpyridin-1-ium-1-yl)hexanoic acid (MP-lysine) as reference material, its amounts were quantitated in acrolein-modified peanut proteins by HPLC-ESI-MS/MS after acid hydrolysis, showing that at low acrolein concentrations, the modification of lysine could be entirely explained by the formation of MP-lysine. Furthermore, for the first time, MP-lysine was quantitated in peanut samples in amounts up to 10.2 mg/kg, showing an increase depending on the roasting time. Thus, MP-lysine might represent a marker to evaluate the extent of food protein lipation by acrolein. PMID:27499313

  13. Kinetics of CO2 Absorption into Aqueous Basic Amino Acid Salt: Potassium Salt of Lysine Solution.

    PubMed

    Shen, Shufeng; Yang, Ya-nan; Bian, Yangyang; Zhao, Yue

    2016-02-16

    Aqueous amino acid salts are considered as an attractive alternative to alkanolamine solvents (e.g., MEA) for carbon dioxide (CO2) absorption. The kinetics of CO2 into unloaded aqueous solutions of potassium lysinate (LysK) was studied using a wetted wall column at concentrations ranging from 0.25 to 2.0 M and temperatures from 298 to 333 K. Physicochemical properties of aqueous LysK solutions such as density, viscosity, and physical solubility of CO2 were measured to evaluate the reaction rate constants. The reaction pathway is described using zwitterion mechanism taking into account the effect of ionic strength on the reaction rate. Under the fast pseudo-first-order regime, the reaction rate parameters were obtained and correlated in a power-law reaction rate expression. LysK shows higher chemical reactivity toward CO2 than the industrial standard MEA and most of amino acid salts. Its reaction rate constants increase considerably with concentration and temperature. The reaction order is found to be an average value of 1.58 with respect to LysK. The forward second-order kinetic rate constant, k2 0 , are obtained as 31615 and 84822 m3 kmol−1 s−1 at 298 and 313 K, respectively with activation energy of 51.0 kJ mol−1. The contribution of water to the zwitterion deprotonation seems to be more significant than that of LysK for the above-mentioned kinetic conditions PMID:26751093

  14. A l-Lysine Transporter of High Stereoselectivity of the Amino Acid-Polyamine-Organocation (APC) Superfamily

    PubMed Central

    Kaur, Jagdeep; Olkhova, Elena; Malviya, Viveka Nand; Grell, Ernst; Michel, Hartmut

    2014-01-01

    Membrane proteins of the amino acid-polyamine-organocation (APC) superfamily transport amino acids and amines across membranes and play an important role in the regulation of cellular processes. We report the heterologous production of the LysP-related transporter STM2200 from Salmonella typhimurium in Escherichia coli, its purification, and functional characterization. STM2200 is assumed to be a proton-dependent APC transporter of l-lysine. The functional interaction between basic amino acids and STM2200 was investigated by thermoanalytical methods, i.e. differential scanning and isothermal titration calorimetry. Binding of l-lysine to STM2200 in its solubilized monomer form is entropy-driven. It is characterized by a dissociation constant of 40 μm at pH 5.9 and is highly selective; no evidence was found for the binding of l-arginine, l-ornithine, l-2,4-diaminobutyric acid, and l-alanine. d-Lysine is bound 45 times more weakly than its l-chiral form. We thus postulate that STM2200 functions as a specific transport protein. Based on the crystal structure of ApcT (Shaffer, P. L., Goehring, A., Shankaranarayanan, A., and Gouaux, E. (2009) Science 325, 1010–1014), a proton-dependent amino acid transporter of the APC superfamily, a homology model of STM2200 was created. Docking studies allowed identification of possible ligand binding sites. The resulting predictions indicated that Glu-222 and Arg-395 of STM2200 are markedly involved in ligand binding, whereas Lys-163 is suggested to be of structural and functional relevance. Selected variants of STM2200 where these three amino acid residues were substituted using single site-directed mutagenesis showed no evidence for l-lysine binding by isothermal titration calorimetry, which confirmed the predictions. Molecular aspects of the observed ligand specificity are discussed. PMID:24257746

  15. Alpha-lipoic acid affects the oxidative stress in various brain structures in mice with methionine and choline deficiency.

    PubMed

    Veskovic, Milena; Mladenovic, Dusan; Jorgacevic, Bojan; Stevanovic, Ivana; de Luka, Silvio; Radosavljevic, Tatjana

    2015-04-01

    Deficiency in methionine or choline can induce oxidative stress in various organs such as liver, kidney, heart, and brain. This study was to examine the effects of alpha-lipoic acid (LA) on oxidative stress induced by methionine and choline deficiency (MCD) in several brain structures. Male mice C57BL/6 (n = 28) were divided into four groups: (1) control - continuously fed with standard chow; (2) LA - fed with standard chow and receiving LA; (3) MCD2 - fed with MCD diet for two weeks, and (4) MCD2+LA - fed with MCD diet for two weeks and receiving LA (100 mg/kg/day intraperitonealy [i.p.]). Brain tissue (cortex, hypothalamus, striatum and hippocampus) was taken for determination of oxidative stress parameters. MCD diet induced a significant increase in malondialdehyde and NOx concentration in all brain regions, while LA restored their content to normal values. Similar to this, in MCD2 group, activity of total SOD, MnSOD, and Cu/ZnSOD was reduced by MCD diet, while LA treatment improved their activities in all brain structures. Besides, in MCD2 group a decrease in catalase activity in cortex and GSH content in hypothalamus was evident, while LA treatment induced an increase in catalase activity in cortex and striatum and GSH content in hypothalamus. LA treatment can significantly reduce lipid peroxidation and nitrosative stress, caused by MCD diet, in all brain regions by restoring antioxidant enzymes activities, predominantly total SOD, MnSOD, and Cu/ZnSOD, and to a lesser extent by modulating catalase activity and GSH content. LA supplementation may be used in order to prevent brain oxidative injury induced by methionine and choline deficiency.

  16. Alpha-lipoic acid affects the oxidative stress in various brain structures in mice with methionine and choline deficiency

    PubMed Central

    Veskovic, Milena; Mladenovic, Dusan; Jorgacevic, Bojan; Stevanovic, Ivana; de Luka, Silvio

    2015-01-01

    Deficiency in methionine or choline can induce oxidative stress in various organs such as liver, kidney, heart, and brain. This study was to examine the effects of alpha-lipoic acid (LA) on oxidative stress induced by methionine and choline deficiency (MCD) in several brain structures. Male mice C57BL/6 (n = 28) were divided into four groups: (1) control – continuously fed with standard chow; (2) LA – fed with standard chow and receiving LA; (3) MCD2 – fed with MCD diet for two weeks, and (4) MCD2+LA – fed with MCD diet for two weeks and receiving LA (100 mg/kg/day intraperitonealy [i.p.]). Brain tissue (cortex, hypothalamus, striatum and hippocampus) was taken for determination of oxidative stress parameters. MCD diet induced a significant increase in malondialdehyde and NOx concentration in all brain regions, while LA restored their content to normal values. Similar to this, in MCD2 group, activity of total SOD, MnSOD, and Cu/ZnSOD was reduced by MCD diet, while LA treatment improved their activities in all brain structures. Besides, in MCD2 group a decrease in catalase activity in cortex and GSH content in hypothalamus was evident, while LA treatment induced an increase in catalase activity in cortex and striatum and GSH content in hypothalamus. LA treatment can significantly reduce lipid peroxidation and nitrosative stress, caused by MCD diet, in all brain regions by restoring antioxidant enzymes activities, predominantly total SOD, MnSOD, and Cu/ZnSOD, and to a lesser extent by modulating catalase activity and GSH content. LA supplementation may be used in order to prevent brain oxidative injury induced by methionine and choline deficiency. PMID:25193852

  17. Mobility of lysozyme in poly(l-lysine)/hyaluronic acid multilayer films.

    PubMed

    Velk, Natalia; Uhlig, Katja; Vikulina, Anna; Duschl, Claus; Volodkin, Dmitry

    2016-11-01

    The spatial and temporal control over presentation of protein-based biomolecules such as growth factors and hormones is crucial for in vitro applications to mimic the complex in vivo environment. We investigated the interaction of a model protein lysozyme (Lys) with poly(L-lysine)/hyaluronic acid (PLL/HA) multilayer films. We focused on Lys diffusion as well as adsorption and retention within the film as a function of the film deposition conditions and post-treatment. Additionally, an effect of Lys concentration on its mobility was probed. A combination of confocal fluorescence microscopy, fluorescence recovery after photobleaching, and microfluidics was employed for this investigation. Our main finding is that adsorption of PLL and HA after protein loading induces acceleration and reduction of Lys mobility, respectively. These results suggest that a charge balance in the film to a high extent governs the protein-film interaction. We believe that control over protein mobility is a key to reach the full potential of the PLL/HA films as reservoirs for biomolecules depending on the application demand. PMID:27552029

  18. Heterologous Production of Hyaluronic Acid in an ε-Poly-l-Lysine Producer, Streptomyces albulus

    PubMed Central

    Yoshimura, Tomohiro; Shibata, Nobuyuki; Hamano, Yoshimitsu

    2015-01-01

    Hyaluronic acid (HA) is used in a wide range of medical applications, where its performance and therapeutic efficacy are highly dependent on its molecular weight. In the microbial production of HA, it has been suggested that a high level of intracellular ATP enhances the productivity and molecular weight of HA. Here, we report on heterologous HA production in an ε-poly-l-lysine producer, Streptomyces albulus, which has the potential to generate ATP at high level. The hasA gene from Streptococcus zooepidemicus, which encodes HA synthase, was refactored and expressed under the control of a late-log growth phase-operating promoter. The expression of the refactored hasA gene, along with genes coding for UDP-glucose dehydrogenase, UDP-N-acetylglucosamine pyrophosphorylase, and UDP-glucose pyrophosphorylase, which are involved in HA precursor sugar biosynthesis, resulted in efficient production of HA in the 2.0 MDa range, which is greater than typical bacterial HA, demonstrating that a sufficient amount of ATP was provided to support the biosynthesis of the precursor sugars, which in turn promoted HA production. In addition, unlike in the case of streptococcal HA, S. albulus-derived HA was not cell associated. Based on these findings, our heterologous production system appears to have several advantages for practical HA production. We propose that the present system could be applicable to the heterologous production of a wide variety of molecules other than HA in the case their biosynthesis pathways require ATP in vivo. PMID:25795665

  19. First hyperpolarizability of the natural aromatic amino acids tryptophan, tyrosine, and phenylalanine and the tripeptide lysine-tryptophan-lysine determined by hyper-Rayleigh scattering.

    PubMed

    Duboisset, J; Matar, G; Russier-Antoine, I; Benichou, E; Bachelier, G; Jonin, Ch; Ficheux, D; Besson, F; Brevet, P F

    2010-11-01

    We report the first hyperpolarizability of tryptophan (Trp) and tyrosine (Tyr) and an upper limit for that of phenylalanine (Phe), three natural aromatic amino acids. The measurements were performed with hyper-Rayleigh scattering in an aqueous Tris buffer solution at a pH of 8.5 and 150 mM salt concentration with a fundamental wavelength of 780 nm. A value of (4.7 ± 0.7) × 10(-30) esu is found for Trp and (4.1 ± 0.7) × 10(-30) esu for Tyr whereas the upper limit of 1.4 × 10(-30) esu is found for that of Phe due to its limited solubility. The influence of the presence of lysine (Lys) in close vicinity of Trp is investigated with a measurement of the first hyperpolarizabilty of Trp in an excess of Lys and compared to the first hyperpolarizability obtained for the tripeptide Lys-Trp-Lys. The clear decrease of the values measured in these two cases indicates that the first hyperpolarizabilty of Trp is very sensitive to its local environment.

  20. Effect of methionine hydroxy analog supplementation on dairy cattle hoof growth and composition.

    PubMed

    Clark, A K; Rakes, A H

    1982-08-01

    Fifty lactating Holstein cows were assigned randomly to one of two treatments, control and control plus approximately 30 g methionine hydroxy analog, and confined on concrete for 11 mo. The control diet consisted of sorghum silage and concentrate fed as a blended ration. Sulfur contents of dry matter were .12% and .16% for control and methionine hydroxy analog rations. Hoof growth and hardness were measured on front and rear right abaxial claws in the dorsal and lateral regions. Hoof growth rates were measured for four periods; summer-fall, fall-winter, winter-spring, and spring-summer, each 70 to 90 days. Hooves of cows fed methionine hydroxy analog grew faster than those of control cows during spring-summer in all regions. Variations of growth rates of hooves were seasonal and tended to follow variations in daily photoperiod. Wear rates were not affected significantly by treatment. Hooves of cows fed methionine hydroxy analog were softer in the top dorsal region at the end of winter-spring and in the dorsal toe region at the end of spring-summer. All other locations were not affected significantly by treatment. The toe region was harder than the top of the hoof. Cows fed methionine hydroxy analog had less cysteine and proline in hoof than control cows and greater percentages of methionine lysine, tyrosine, and glutamic acid. These results suggest that a decrease of disulfide bonding occurred in the hoof tissue of cows fed methionine hydroxy analog. Cows fed methionine hydroxy analog produced more actual milk, milk fat, and 4% fat-corrected milk during 180 days than did control cows. PMID:6183300

  1. Effect of methionine hydroxy analog supplementation on dairy cattle hoof growth and composition.

    PubMed

    Clark, A K; Rakes, A H

    1982-08-01

    Fifty lactating Holstein cows were assigned randomly to one of two treatments, control and control plus approximately 30 g methionine hydroxy analog, and confined on concrete for 11 mo. The control diet consisted of sorghum silage and concentrate fed as a blended ration. Sulfur contents of dry matter were .12% and .16% for control and methionine hydroxy analog rations. Hoof growth and hardness were measured on front and rear right abaxial claws in the dorsal and lateral regions. Hoof growth rates were measured for four periods; summer-fall, fall-winter, winter-spring, and spring-summer, each 70 to 90 days. Hooves of cows fed methionine hydroxy analog grew faster than those of control cows during spring-summer in all regions. Variations of growth rates of hooves were seasonal and tended to follow variations in daily photoperiod. Wear rates were not affected significantly by treatment. Hooves of cows fed methionine hydroxy analog were softer in the top dorsal region at the end of winter-spring and in the dorsal toe region at the end of spring-summer. All other locations were not affected significantly by treatment. The toe region was harder than the top of the hoof. Cows fed methionine hydroxy analog had less cysteine and proline in hoof than control cows and greater percentages of methionine lysine, tyrosine, and glutamic acid. These results suggest that a decrease of disulfide bonding occurred in the hoof tissue of cows fed methionine hydroxy analog. Cows fed methionine hydroxy analog produced more actual milk, milk fat, and 4% fat-corrected milk during 180 days than did control cows.

  2. Interaction of genetic mechanisms regulating methionine concentration in maize grain

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Methionine is a limiting amino acid in poultry diets so methionine supplementation is typically required to meet nutritional demands. Maize varieties with increased methionine levels have been developed utilizing three different approaches: i.) increased levels of the methionine-rich 10 kDa zein, ii...

  3. Lysine biosynthesis and nitrogen metabolism in quinoa (Chenopodium quinoa): study of enzymes and nitrogen-containing compounds.

    PubMed

    Varisi, Vanderlei A; Camargos, Liliane S; Aguiar, Leandro F; Christofoleti, Renata M; Medici, Leonardo O; Azevedo, Ricardo A

    2008-01-01

    Aspartate kinase (AK, EC 2.7.2.4), homoserine dehydrogenase (HSDH, EC 1.1.1.3) and dihydrodipicolinate synthase (DHDPS, EC 4.2.1.52) were isolated and partially purified from immature Chenopodium quinoa Willd seeds. Enzyme activities were studied in the presence of the aspartate-derived amino acids lysine, threonine and methionine and also the lysine analogue S-2-aminoethyl-l-cysteine (AEC), at 1 mM and 5 mM. The results confirmed the existence of, at least, two AK isoenzymes, one inhibited by lysine and the other inhibited by threonine, the latter being predominant in quinoa seeds. HSDH activity was also shown to be partially inhibited by threonine, whereas some of the activity was resistant to the inhibitory effect, indicating the presence of two isoenzymes, one resistant and another sensitive to threonine inhibition. Only one DHDPS isoenzyme highly sensitive to lysine inhibition was detected. The results suggest that the high concentration of lysine observed in quinoa seeds is possibly due to a combined effect of increased lysine synthesis and accumulation in the soluble form and/or as protein lysine. Nitrogen assimilation was also investigated and based on nitrate content, nitrate reductase activity, amino acid distribution and ureide content, the leaves were identified as the predominant site of nitrate reduction in this plant species. The amino acid profile analysis in leaves and roots also indicated an important role of soluble glutamine as a nitrogen transporting compound.

  4. Biosynthesis of pipecolic acid by RapL, a lysine cyclodeaminase encoded in the rapamycin gene cluster.

    PubMed

    Gatto, Gregory J; Boyne, Michael T; Kelleher, Neil L; Walsh, Christopher T

    2006-03-22

    Rapamycin, FK506, and FK520 are immunosuppressant macrolactone natural products comprised of predominantly polyketide-based core structures. A single nonproteinogenic pipecolic acid residue is installed into the scaffold by a nonribosomal peptide synthetase that also performs the subsequent macrocyclization step at the carbonyl group of this amino acid. It has been assumed that pipecolic acid is generated from lysine by the cyclodeaminases RapL/FkbL. Herein we report the heterologous overexpression and purification of RapL and validate its ability to convert L-lysine to L-pipecolic acid by a cyclodeamination reaction that involves redox catalysis. RapL also accepts L-ornithine as a substrate, albeit with a significantly reduced catalytic efficiency. Turnover is presumed to encompass a reversible oxidation at the alpha-amine, internal cyclization, and subsequent re-reduction of the cyclic delta1-piperideine-2-carboxylate intermediate. As isolated, RapL has about 0.17 equiv of tightly bound NAD+, suggesting that the enzyme is incompletely loaded when overproduced in E. coli. In the presence of exogenous NAD+, the initial rate is elevated 8-fold with a Km of 2.3 microM for the cofactor, consistent with some release and rebinding of NAD+ during catalytic cycles. Through the use of isotopically labeled substrates, we have confirmed mechanistic details of the cyclodeaminase reaction, including loss of the alpha-amine and retention of the hydrogen atom at the alpha-carbon. In addition to the characterization of a critical enzyme in the biosynthesis of a medically important class of natural products, this work represents the first in vitro characterization of a lysine cyclodeaminase, a member of a unique group of enzymes which utilize the nicotinamide cofactor in a catalytic manner. PMID:16536560

  5. Lysine-functionalized nanodiamonds: synthesis, physiochemical characterization, and nucleic acid binding studies

    PubMed Central

    Kaur, Randeep; Chitanda, Jackson M; Michel, Deborah; Maley, Jason; Borondics, Ferenc; Yang, Peng; Verrall, Ronald E; Badea, Ildiko

    2012-01-01

    Purpose: Detonation nanodiamonds (NDs) are carbon-based nanomaterials that, because of their size (4–5 nm), stable inert core, alterable surface chemistry, fluorescence, and biocompatibility, are emerging as bioimaging agents and promising tools for the delivery of biochemical molecules into cellular systems. However, diamond particles possess a strong propensity to aggregate in liquid formulation media, restricting their applicability in biomedical sciences. Here, the authors describe the covalent functionalization of NDs with lysine in an attempt to develop nanoparticles able to act as suitable nonviral vectors for transferring genetic materials across cellular membranes. Methods: NDs were oxidized and functionalized by binding lysine moieties attached to a three-carbon-length linker (1,3-diaminopropane) to their surfaces through amide bonds. Raman and Fourier transform infrared spectroscopy, zeta potential measurement, dynamic light scattering, atomic force microscopic imaging, and thermogravimetric analysis were used to characterize the lysine-functionalized NDs. Finally, the ability of the functionalized diamonds to bind plasmid DNA and small interfering RNA was investigated by gel electrophoresis assay and through size and zeta potential measurements. Results: NDs were successfully functionalized with the lysine linker, producing surface loading of 1.7 mmol g−1 of ND. These modified NDs formed highly stable aqueous dispersions with a zeta potential of 49 mV and particle size of approximately 20 nm. The functionalized NDs were found to be able to bind plasmid DNA and small interfering RNA by forming nanosized “diamoplexes”. Conclusion: The lysine-substituted ND particles generated in this study exhibit stable aqueous formulations and show potential for use as carriers for genetic materials. PMID:22904623

  6. Monocarboxylate transporter 1 is up-regulated in Caco-2 cells by the methionine precursor DL-2-hydroxy-(4-methylthio)butanoic acid.

    PubMed

    Martín-Venegas, Raquel; Brufau, M Teresa; Mañas-Cano, Oriol; Mercier, Yves; Nonis, Magalie K; Ferrer, Ruth

    2014-12-01

    The methionine precursor, DL-2-hydroxy-(4-methylthio)butanoic acid (HMTBA), is a synthetic source of dietary methionine, which is widely used as a poultry nutritional supplement. In the intestinal epithelium, HMTBA transport across the apical membrane is mediated by monocarboxylate transporter 1 (MCT1). The first step in biological utilisation of this methionine precursor is the stereospecific conversion of HMTBA to the corresponding keto acid. In the present study, the regulation of trans-epithelial HMTBA transport was investigated in Caco-2 cell monolayers. Differentiated Caco-2 cells were maintained under control conditions (apical compartment: 0.2 mmol/L L-methionine) or in a HMTBA-enriched medium (2 mmol/L HMTBA). The effect of culture on HMTBA transport was evaluated from apical and basolateral kinetic parameters. MCT1 and MCT4 immuno-localisation and gene expression were investigated by confocal microscopy and real-time quantitative RT-PCR, respectively. The results indicated that apical MCT1 was up-regulated by exposure to HMTBA (1.4-fold increase in Vmax without changes in Km). Moreover, total monolayer MCT1 immunoreactivity increased 1.8-fold in HMTBA-supplemented cultures, this effect mainly being localised at the apical membrane. Functional and immuno-localisation data suggest involvement of MCT1 and MCT4 in basolateral HMTBA transport, although, in this case, no effect was observed for HMTBA-enrichment. Molecular analysis confirmed MCT1 mRNA up-regulation (1.8-fold), with no effect on MCT4 mRNA expression. Thus, exposure to HMTBA up-regulates the trans-epithelial transport of this methionine precursor by increasing the expression and the transport capacity of apical MCT1. PMID:25447800

  7. Monocarboxylate transporter 1 is up-regulated in Caco-2 cells by the methionine precursor DL-2-hydroxy-(4-methylthio)butanoic acid.

    PubMed

    Martín-Venegas, Raquel; Brufau, M Teresa; Mañas-Cano, Oriol; Mercier, Yves; Nonis, Magalie K; Ferrer, Ruth

    2014-12-01

    The methionine precursor, DL-2-hydroxy-(4-methylthio)butanoic acid (HMTBA), is a synthetic source of dietary methionine, which is widely used as a poultry nutritional supplement. In the intestinal epithelium, HMTBA transport across the apical membrane is mediated by monocarboxylate transporter 1 (MCT1). The first step in biological utilisation of this methionine precursor is the stereospecific conversion of HMTBA to the corresponding keto acid. In the present study, the regulation of trans-epithelial HMTBA transport was investigated in Caco-2 cell monolayers. Differentiated Caco-2 cells were maintained under control conditions (apical compartment: 0.2 mmol/L L-methionine) or in a HMTBA-enriched medium (2 mmol/L HMTBA). The effect of culture on HMTBA transport was evaluated from apical and basolateral kinetic parameters. MCT1 and MCT4 immuno-localisation and gene expression were investigated by confocal microscopy and real-time quantitative RT-PCR, respectively. The results indicated that apical MCT1 was up-regulated by exposure to HMTBA (1.4-fold increase in Vmax without changes in Km). Moreover, total monolayer MCT1 immunoreactivity increased 1.8-fold in HMTBA-supplemented cultures, this effect mainly being localised at the apical membrane. Functional and immuno-localisation data suggest involvement of MCT1 and MCT4 in basolateral HMTBA transport, although, in this case, no effect was observed for HMTBA-enrichment. Molecular analysis confirmed MCT1 mRNA up-regulation (1.8-fold), with no effect on MCT4 mRNA expression. Thus, exposure to HMTBA up-regulates the trans-epithelial transport of this methionine precursor by increasing the expression and the transport capacity of apical MCT1.

  8. Differences in plasma metabolomics between sows fed DL-methionine and its hydroxy analogue reveal a strong association of milk composition and neonatal growth with maternal methionine nutrition.

    PubMed

    Zhang, Xiaoling; Li, Hao; Liu, Guangmang; Wan, Haifeng; Mercier, Yves; Wu, Caimei; Wu, Xiuqun; Che, Lianqiang; Lin, Yan; Xu, Shengyu; Tian, Gang; Chen, Daiwen; Wu, De; Fang, Zhengfeng

    2015-02-28

    The aim of the present study was to determine whether increased consumption of methionine as DL-methionine (DLM) or its hydroxy analogue DL-2-hydroxy-4-methylthiobutanoic acid (HMTBA) could benefit milk synthesis and neonatal growth. For this purpose, eighteen cross-bred (Landrace × Yorkshire) primiparous sows were fed a control (CON), DLM or HMTBA diet (n 6 per diet) from 0 to 14 d post-partum. At postnatal day 14, piglets in the HMTBA group had higher body weight (P= 0·02) than those in the CON group, tended (P= 0·07) to be higher than those in the DLM group, and had higher (P< 0·05) mRNA abundance of jejunal fatty acid-binding protein 2, intestinal than those in the CON and DLM groups. Compared with the CON diet-fed sows, milk protein, non-fat solid, and lysine, histidine and ornithine concentrations decreased in the DLM diet-fed sows (P< 0·05), and milk fat, lactose, and cysteine and taurine concentrations increased in the HMTBA diet-fed sows (P< 0·05). Plasma homocysteine and urea N concentrations that averaged across time were increased (P< 0·05) in sows fed the DLM diet compared with those fed the CON diet. Metabolomic results based on ¹H NMR spectroscopy revealed that consumption of the HMTBA and DLM diets increased (P< 0·05) both sow plasma methionine and valine levels; however, consumption of the DLM diet led to lower (P< 0·05) plasma levels of lysine, tyrosine, glucose and acetate and higher (P< 0·05) plasma levels of citrate, lactate, formate, glycerol, myo-inositol and N-acetyl glycoprotein in sows. Collectively, neonatal growth and milk synthesis were regulated by dietary methionine levels and sources, which resulted in marked alterations in amino acid, lipid and glycogen metabolism.

  9. Acetylmethionine as a source of methionine for the rat.

    PubMed

    Boggs, R W; Rotruck, J T; Damico, R A

    1975-03-01

    A-Acetyl-L-methionine and N-acetyl-D-methionine were compared with L-methionine and D-methionine as sources of methionine. These derivatives were added to a sulfer amino acid-limited diet containing 10% soybean protein isolate. Weight gains, food intake, and protein efficiency ratios (PER) were determined in growing rats. N-Acetyl-L-methionine, L-methionine, and D-methionine produced an equivalent growth response and increase in PER above that of the basal diet. There was no response to supplementation with N-acetyl-D-methionine. An equivalent maximum growth response of rats fed-L-methionine or N-acetyl-D-methionine. An equivalent maximum growth response of rats fed L-methionine or N-acetyl-L-methionine was obtained when the total dietary sulfur amino acids compromised 0.36-0.41% of the diet. The nutritional similarities of methionine and N-acetyl-L-methionine suggest that the latter may be useful as a supplement to diets containing vegetable proteins that are deficient in sulfur amino acids.

  10. Mycobacterium Lysine ε-aminotransferase is a novel alarmone metabolism related persister gene via dysregulating the intracellular amino acid level.

    PubMed

    Duan, Xiangke; Li, Yunsong; Du, Qinglin; Huang, Qinqin; Guo, Siyao; Xu, Mengmeng; Lin, Yanping; Liu, Zhidong; Xie, Jianping

    2016-01-01

    Bacterial persisters, usually slow-growing, non-replicating cells highly tolerant to antibiotics, play a crucial role contributing to the recalcitrance of chronic infections and treatment failure. Understanding the molecular mechanism of persister cells formation and maintenance would obviously inspire the discovery of new antibiotics. The significant upregulation of Mycobacterium tuberculosis Rv3290c, a highly conserved mycobacterial lysine ε-aminotransferase (LAT) during hypoxia persistent model, suggested a role of LAT in persistence. To test this, a lat deleted Mycobacterium smegmatis was constructed. The expression of transcriptional regulator leucine-responsive regulatory protein (LrpA) and the amino acids abundance in M. smegmatis lat deletion mutants were lowered. Thus, the persistence capacity of the deletion mutant was impaired upon norfloxacin exposure under nutrient starvation. In summary, our study firstly reported the involvement of mycobacterium LAT in persister formation, and possibly through altering the intracellular amino acid metabolism balance. PMID:26806099

  11. The Coding Properties of Lysine-accepting Transfer Ribonucleic Acids from Black-eyed Peas 1

    PubMed Central

    Hague, Donald R.; Kofoid, Eric C.

    1971-01-01

    Lysine-accepting transfer RNA from ungerminated and germinated embryo axes of black-eyed peas (Vigna sinensis L. Savi) was fractionated on benzoylated diethylaminoethyl cellulose and reverse phase Freon columns. Cochromatography indicated the presence of two similar lysyl transfer RNA fractions in each tissue. Ribosome binding studies revealed that the larger of the two fractions in each case is specific for the AAG codon, while the smaller one recognizes AAA and AAG. Possible implications of this difference in quantities of isoacceptors in translation of genetic information are discussed. PMID:16657787

  12. The Coding Properties of Lysine-accepting Transfer Ribonucleic Acids from Black-eyed Peas.

    PubMed

    Hague, D R; Kofoid, E C

    1971-09-01

    Lysine-accepting transfer RNA from ungerminated and germinated embryo axes of black-eyed peas (Vigna sinensis L. Savi) was fractionated on benzoylated diethylaminoethyl cellulose and reverse phase Freon columns. Cochromatography indicated the presence of two similar lysyl transfer RNA fractions in each tissue. Ribosome binding studies revealed that the larger of the two fractions in each case is specific for the AAG codon, while the smaller one recognizes AAA and AAG. Possible implications of this difference in quantities of isoacceptors in translation of genetic information are discussed.

  13. The lysine-ketoglutarate reductase-saccharopine dehydrogenase is involved in the osmo-induced synthesis of pipecolic acid in rapeseed leaf tissues.

    PubMed

    Moulin, M; Deleu, C; Larher, F; Bouchereau, A

    2006-01-01

    Higher plant responses to abiotic stresses are associated with physiological and biochemical changes triggering a number of metabolic adjustments. We focused on L-lysine catabolism, and have previously demonstrated that degradation of this amino acid is osmo-regulated at the level of lysine-ketoglutarate reductase (LKR, EC 1.5.1.8) and saccharopine dehydrogenase (SDH, EC 1.5.1.9) in Brassica napus. LKR and SDH activities are enhanced by decreasing osmotic potential and decrease when the upshock osmotic treatment is followed by a downshock osmotic one. Moreover we have shown that the B. napus LKR/SDH gene is up-regulated in osmotically-stressed tissues. The LKR/SDH activity produces alpha-aminoadipate semialdehyde which could be further converted into alpha-aminoadipate and acetyl CoA. Alternatively alpha-aminoadipate could behave as a precursor for pipecolic acid. Pipecolic acid is described as an osmoprotectant in bacteria and is co-accumulated with proline in halophytic plants. We suggest that osmo-induction of the LKR/SDH activity could be partly responsible for pipecolic acid accumulation. This proposal has been assessed in this study through pipecolic acid amounts determination in rape leaf discs subjected to various upshift and downshift osmotic treatments. Changes in pipecolic acid level actually behave as those observed for LKR and SDH activities, since it increases or decreases in rape leaf discs treated under hyper- or hypo-osmotic conditions, respectively. In addition we show that pipecolic acid level is positively correlated with the external osmotic potential as well as with the duration of the applied treatment. On the other hand pipecolic acid level is related to the availability of L-lysine and not to that of D-lysine. Collectively the results obtained demonstrate that lysine catabolism through LKR/SDH activity is involved in osmo-induced synthesis of pipecolic acid.

  14. Preparation, characterization, and biocompatibility evaluation of poly(Nɛ-acryloyl-L-lysine)/hyaluronic acid interpenetrating network hydrogels.

    PubMed

    Cui, Ning; Qian, Junmin; Xu, Weijun; Xu, Minghui; Zhao, Na; Liu, Ting; Wang, Hongjie

    2016-01-20

    In the present study, poly(Nɛ-acryloyl-L-lysine)/hyaluronic acid (pLysAAm/HA) interpenetrating network (IPN) hydrogels were successfully fabricated through the combination of hydrazone bond crosslinking and photo-crosslinking reactions. The HA hydrogel network was first synthesized from 3,3'-dithiodipropionate hydrazide-modified HA and polyethylene glycol dilevulinate by hydrazone bond crosslinking. The pLysAAm hydrogel network was prepared from Nɛ-acryloyl-L-lysine and N,N'-bis(acryloyl)-(L)-cystine by photo-crosslinking. The resultant pLysAAm/HA hydrogels had a good shape recovery property after loading and unloading for 1.5 cycles (up to 90%) and displayed a highly porous microstructure. Their compressive moduli were at least 5 times higher than that of HA hydrogels. The pLysAAm/HA hydrogels had an equilibrium swelling ratio of up to 37.9 and displayed a glutathione-responsive degradation behavior. The results from in vitro biocompatibility evaluation with pre-osteoblasts MC3T3-E1 cells revealed that the pLysAAm/HA hydrogels could support cell viability and proliferation. Hematoxylin and eosin staining indicated that the pLysAAm/HA hydrogels allowed cell and tissue infiltration, confirming their good in vivo biocompatibility. Therefore, the novel pLysAAm/HA IPN hydrogels have great potential for bone tissue engineering applications.

  15. Inhibition of corneal neovascularization with a nutrient mixture containing lysine, proline, ascorbic acid, and green tea extract.

    PubMed

    Shakiba, Yadollah; Mostafaie, Ali

    2007-10-01

    Corneal neovascularization is a significant, sight-threatening complication of many ocular surface disorders. Various growth factors and proteinases are involved in corneal neovascularization. The data supporting a causal role for vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs) are extensive. Inhibition of VEGF and MMPs is a main strategy for treating corneal neovascularization. Several findings have shown that corneal neovascularization can be reduced by using anti-VEGF and anti-MMPs agents. Efficacy of a nutrient mixture (NM) containing lysine, proline, ascorbic acid, and green tea extract has been demonstrated for reducing VEGF and MMPs secretion by various cells. Moreover, NM can inhibit endothelial cell migration and capillary tube formation. We herein note that topical application of NM is potentially useful for inhibiting corneal neovascularization and restoration of corneal clarity. Further investigations in animal models are needed to place NM alongside corneal neovascularization therapeutics.

  16. Quantitation of cellular metabolic fluxes of methionine.

    PubMed

    Shlomi, Tomer; Fan, Jing; Tang, Baiqing; Kruger, Warren D; Rabinowitz, Joshua D

    2014-02-01

    Methionine is an essential proteogenic amino acid. In addition, it is a methyl donor for DNA and protein methylation and a propylamine donor for polyamine biosynthesis. Both the methyl and propylamine donation pathways involve metabolic cycles, and methods are needed to quantitate these cycles. Here, we describe an analytical approach for quantifying methionine metabolic fluxes that accounts for the mixing of intracellular and extracellular methionine pools. We observe that such mixing prevents isotope tracing experiments from reaching the steady state due to the large size of the media pools and hence precludes the use of standard stationary metabolic flux analysis. Our approach is based on feeding cells with (13)C methionine and measuring the isotope-labeling kinetics of both intracellular and extracellular methionine by liquid chromatography-mass spectrometry (LC-MS). We apply this method to quantify methionine metabolism in a human fibrosarcoma cell line and study how methionine salvage pathway enzyme methylthioadenosine phosphorylase (MTAP), frequently deleted in cancer, affects methionine metabolism. We find that both transmethylation and propylamine transfer fluxes amount to roughly 15% of the net methionine uptake, with no major changes due to MTAP deletion. Our method further enables the quantification of flux through the pro-tumorigenic enzyme ornithine decarboxylase, and this flux increases 2-fold following MTAP deletion. The analytical approach used to quantify methionine metabolic fluxes is applicable for other metabolic systems affected by mixing of intracellular and extracellular metabolite pools.

  17. Nutritional consequences of the reactions between proteins and oxidized polyphenolic acids.

    PubMed

    Hurrell, R F; Finot, P A

    1984-01-01

    The chemical and enzymatic browning reactions of plant polyphenols and their effects on amino acids and proteins are reviewed. A model system of casein and oxidizing caffeic acid has been studied in more detail. The effects of pH, time, caffeic acid level and the presence or not of tyrosinase on the decrease of FDNB-reactive lysine are described. The chemical loss of lysine, methionine and tryptophan and the change in the bioavailability of these amino acids to rats has been evaluated in two systems: pH 7.0 with tyrosinase and pH 10.0 without tyrosinase. At pH 10.0, reactive lysine was more reduced. At pH 7.0 plus tyrosinase methionine was more extensively oxidized to its sulphoxide. Tryptophan was not chemically reduced under either condition. At pH 10.0 there was a decrease in the protein digestibility which was responsible for a corresponding reduction in tryptophan availability and partly responsible for lower methionine availability. Metabolic transit of casein labelled with tritiated lysine treated under the same conditions indicated that the lower lysine availability in rats was due to a lower digestibility of the lysine-caffeoquinone complexes. PMID:6496220

  18. Effect of L- or DL-methionine Supplementation on Nitrogen Retention, Serum Amino Acid Concentrations and Blood Metabolites Profile in Starter Pigs

    PubMed Central

    Tian, Q. Y.; Zeng, Z. K.; Zhang, Y. X.; Long, S. F.; Piao, X. S.

    2016-01-01

    The objective of the current study was to evaluate the effect of supplementation of either L-methionine (L-Met) or DL-methionine (DL-Met) to diets of starter pigs on nitrogen (N) balance, metabolism, and serum amino acid profile. Eighteen crossbred (Duroc×Landrace×Yorkshire) barrows weighing 15.45±0.88 kg were randomly allotted to 1 of 3 diets with 6 pigs per treatment. The diets included a basal diet (Met-deficient diet) containing 0.24% standardized ileal digestibility Met with all other essential nutrients meeting the pig’s requirements. The other two diets were produced by supplementing the basal diet with 0.12% DL-Met or L-Met. The experiment lasted for 18 days, consisting of a 13-day adaptation period to the diets followed by a 5-day experimental period. Pigs were fed ad libitum and free access to water throughout the experiment. Results showed that the supplementation of either L-Met or DL-Met improved N retention, and serum methionine concentration, and decreased N excretion compared with basal diet (p<0.01). The N retention of pigs fed diets supplemented with the same inclusion levels of DL-Met or L-Met were not different (p>0.05). In conclusion, on equimolar basis DL-Met and L-Met are equally bioavailable as Met sources for starter pigs. PMID:26954214

  19. Elimination of amino acids in acute renal failure.

    PubMed

    Druml, W; Bürger, U; Kleinberger, G; Lenz, K; Laggner, A

    1986-01-01

    Plasma amino acid concentrations and the elimination of parenterally administered amino acids were investigated in 12 patients with nonhypercatabolic acute renal failure. A distinctive plasma amino acid pattern could be observed: plasma concentrations of phenylalanine and methionine were increased, those of valine and leucine decreased. Of the nonessential amino acids, cystine, taurine und tyrosine had elevated but none of them reduced plasma concentrations. The elimination of amino acids was evaluated in a monocompartment model after bolus injection of an amino acid solution containing essential and nonessential amino acids. Pharmacokinetic parameters of 17 amino acids were calculated. The mean elimination half-time was raised by 25%. The elimination half-time of phenylalanine, methionine, glutamic acid, proline and ornithine was increased. Histidine was the only amino acid with--however insignificantly--accelerated elimination from the intravascular compartment. The total clearance rate and total transfer rate was not altered (107 and 97% of normal, respectively). The clearance of threonine, lysine, serine, glycine and histidine was increased, of valine, phenylalanine, glutamic acid and to a minor degree of methionine was decreased. The transfer rate of methionine, lysine, glycine was elevated, of valine, aspartic acid, glutamic acid and ornithine reduced. The demonstration of these pronounced alterations of amino acid elimination in acute renal failure may have major consequences in parenteral amino acid therapy.

  20. Methionine-sensitive glycolysis in transformed cells.

    PubMed

    Boerner, P; Racker, E

    1985-10-01

    Glycolysis in several tumor cell lines grown in tissue culture was inhibited by methionine. Kirsten murine sarcoma virus-transformed rat kidney cells (K-NRK) were inhibited 60-75% by 10 mM methionine, whereas normal rat kidney (NRK-49F) cells showed little or no inhibition. Inhibition of glycolysis in K-NRK cells was manifest 2-4 hr after exposure to the amino acid. Glycolysis in a chemically transformed cell line of Madin-Darby canine kidney cells was also sensitive to methionine, but maximal inhibition (75%) required 18-24 hr of incubation with the amino acid. Under the same conditions glycolysis in the nontransformed canine cells was less than 20% inhibited by methionine. In Ehrlich ascites tumor cells grown in tissue culture, 10 mM methionine inhibited glycolysis by about 50%. Inhibition of glycolysis, even by 50 mM methionine, was rapidly reversible. Within 2 hr after removal of methionine the rate of glycolytic activity was restored to that observed in control cells. Furthermore, inhibition by methionine required a minimum level (7%) of serum in the growth medium and inhibition was not sensitive to cycloheximide. Only amino acids that are transported by system A (including the nonmetabolized analogue methylaminoisobutyric acid) specifically inhibited glycolysis in tumor cells. The only exception was phenylalanine, which was toxic to both transformed and normal cell lines.

  1. [The mechanism of potentiation of the antitumor effect of 5-fluorouracil by methionine-free intravenous amino acid solution (AO-90) in rats].

    PubMed

    Hibino, Y; Kawarabayashi, Y; Kohri, H; Ueda, N; Tsukagoshi, S

    1994-09-01

    AO-90, a methionine-free intravenous amino acid solution (7.43%) showed to potentiate the antitumor effect of 5-fluorouracil (5-FU) when concomitantly used as the nitrogen source in total parenteral nutrition (TPN) in Yoshida sarcoma (YS)-bearing rats. In the present experiment, this potentiation mechanism was studied by determining the serum methionine level and tumor methylenetetrahydrofolate (CH2FH4) content in YS-bearing Donryu rats given AO-90 (nitrogen 0.73g/kg on the 1st day and 1.46g/kg for the remaining 6 days) by TPN for 1 week. The rats were subcutaneously inoculated with 10(4) YS cells in the dorsum 3 days before the start of TPN. Inhibition of thymidylate synthase activity in tumor tissue after dosing of AO-90 (nitrogen 0.68g/kg on the 1st day and 1.36 g/kg for the remaining 6 days) by TPN along with daily intraperitoneal dosing of 5-FU (10 mg/kg) was also evaluated with the inoculation of 10(6) tumor cells. The results were compared with those in tumor-bearing rats given TPN with a commercially available amino acid solution containing methionine. On day 5 of TPN, the tumor-bearing rats given AO-90 showed a significantly lower serum methionine level than the control rats: 101 +/- 11 mumol/l versus 29 +/- 14 mumol/l (p < 0.01); and a higher CH2FH4 content in tumor: 7.0 +/- 2.8 pmol/g protein versus 23.7 +/- 16.6 pmol/g protein (p < 0.05). Thymidylate synthase inhibition was 81.2 +/- 5.1% in the AO-90 group and 30.9 +/- 26.3% in the control group (p < 0.01). The results of the present study suggest that AO-90 potentiate the antitumor effect of 5-FU by biochemical modulation. AO-90 concomitantly given with 5-FU for 7 days was effective not only in the allogeneic tumor model, but also in WKAH and SHR rats previously inoculated with 10(6) of syngeneic KDH-8 hepatoma cells and SST-2 adenocarcinoma cells, respectively. Weight of SST-2 adenocarcinoma in SHR rats after the TPN period was significantly smaller in the AO-90 group than in the control rats given

  2. Peptide nucleic acids tagged with four lysine residues for amperometric genosensors

    PubMed Central

    Zanardi, Chiara; Terzi, Fabio; Seeber, Renato; Baldoli, Clara; Licandro, Emanuela; Maiorana, Stefano

    2012-01-01

    A homothymine PNA decamer bearing four lysine residues has been synthesized as a probe for the development of amperometric sensors. On one hand, the four amino groups introduced make this derivative nine times more soluble than the corresponding homothymine PNA decamer and, on the other hand, allow the stable anchoring of this molecule on Au nanostructured surface through the terminal -NH2 moieties. In particular, XPS and electrochemical investigations performed with hexylamine, as a model molecule, indicate that the stable deposition of primary amine derivatives on such a nanostructured surface is possible and involves the free electron doublet on the nitrogen atom. This finding indicates that this PNA derivative is suitable to act as the probe molecule for the development of amperometric sensors.   Thanks to the molecular probe chosen and to the use of a nanostructured surface as the substrate for the sensor assembly, the device proposed makes possible the selective recognition of the target oligonucleotide sequence with very high sensitivity. PMID:22772036

  3. Oxidation of methionine in PrP is dependent upon the oxidant and the amino acid two positions removed

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background/Introduction. Methionine oxidation has been shown both to be associated with prion formation and implicated in the inhibition of amyloid formation in model systems. This work is based on model systems where hydrogen peroxide was used as an oxidant. Materials and Methods. We developed...

  4. Poly(L-diaminopropionic acid), a novel non-proteinic amino acid oligomer co-produced with poly(ε-L-lysine) by Streptomyces albulus PD-1.

    PubMed

    Xia, Jun; Xu, Hong; Feng, Xiaohai; Xu, Zhaoxian; Chi, Bo

    2013-09-01

    Poly(ε-L-lysine) (ε-PL) producer strain Streptomyces albulus PD-1 secreted a novel polymeric substance into its culture broth along with ε-PL. The polymeric substance was purified to homogeneity and identified. Matrix-assisted laser desorption ionization-time of flight mass spectrometry and nuclear magnetic resonance spectroscopy as well as other analytical techniques revealed that the substance was poly(L-diaminopropionic acid) (PDAP). PDAP is an L-α,β-diaminopropionic acid oligomer linking between amino and carboxylic acid functional groups. The molecular weight of PDAP ranged from 500 to 1500 Da, and no co-polymers composed of L-diaminopropionic acid and L-lysine were present in the culture broth. Compared with ε-PL, PDAP exhibited stronger inhibitory activities against yeasts but weaker activities against bacteria. ε-PL and PDAP co-production was also investigated. Both ε-PL and PDAP were synthesized during the stationary phase of growth, and the final ε-PL and PDAP concentration reached 21.7 and 4.8 g L(-1), respectively, in fed-batch fermentation. Citric acid feeding resulted in a maximum ε-PL concentration of 26.1 g L(-1) and a decrease in the final concentration of PDAP to 3.8 g L(-1). No studies on ε-PL and PDAP co-production in Streptomyces albulus have been reported previously, and inhibition of by-products such as PDAP is potentially useful in ε-PL production. PMID:23775267

  5. L-Methionine adsorption on Cu(110), binding and geometry of the amino acid as a function of coverage

    NASA Astrophysics Data System (ADS)

    Méthivier, Christophe; Humblot, Vincent; Pradier, Claire-Marie

    2015-02-01

    The adsorption of L-methionine on Cu(110) has been characterized by combining in situ Polarization Modulation Infrared Reflection Absorption Spectroscopy (PM-IRRAS) and X-ray Photoelectron Spectroscopy (XPS). Both the chemical state of the molecule, and its anchoring points were determined at various coverage values. Adsorbed methionine is anionic and first interacts with the copper surface via its sulfur and/or oxygen atoms, likely lying flat on the surface; at higher coverage, a stronger interaction of oxygen and nitrogen atoms with copper, evidenced by slight shifts of the XPS peaks, together with an angular dependence of the peak ratios, suggests that the molecule stands up on the surface, interacting with the surface via the N and O atoms but almost not anymore via its S atom. Last but not least, no multilayers were evidenced, and this was explained by the geometry of the molecules which leaves no groups accessible for intermolecular interactions.

  6. FXR-induced lysine-specific histone demethylase, LSD1, reduces hepatic bile acid levels and protects the liver against bile acid toxicity

    PubMed Central

    Kim, Young-Chae; Fang, Sungsoon; Byun, Sangwon; Seok, Sunmi; Kemper, Byron; Kemper, Jongsook Kim

    2015-01-01

    Bile acids (BAs) function as endocrine signaling molecules that activate multiple nuclear and membrane receptor signaling pathways to control fed-state metabolism. Since the detergent-like property of BAs causes liver damage at high concentrations, hepatic BA levels must be tightly regulated. BA homeostasis is regulated largely at the level of transcription by nuclear receptors, particularly the primary bile acid receptor, farnesoid X receptor (FXR), and small heterodimer partner (SHP) that inhibits BA synthesis by recruiting repressive histone-modifying enzymes. Although histone modifiers have been shown to regulate BA-responsive genes, their in vivo functions remain unclear. Here we show that lysine-specific histone demethylase1 (LSD1) is directly induced by BA-activated FXR, is recruited to BA synthetic genes, Cyp7a1 and Cyp8b1, and the BA uptake transporter gene, Ntcp, and removes a gene-activation mark, tri-methylated histone H3 lysine-4, leading to gene repression. LSD1 recruitment was dependent on SHP, and LSD1-mediated demethylation of H3K4-me3 was required for additional repressive histone modifications, H3K9/K14 deacetylation and H3K9 methylation. BA overload, feeding 0.5% cholic acid chow for 6 days, resulted in adaptive responses of altered expression of hepatic genes involved in BA synthesis, transport, and detoxification/conjugation. In contrast, adenoviral-mediated downregulation of hepatic LSD1 blunted these responses, which led to substantial increases in liver and serum BA levels, serum AST/ALT levels, and hepatic inflammation. This study identifies LSD1 as a novel histone-modifying enzyme in the orchestrated regulation mediated by the FXR and SHP that reduces hepatic BA levels and protects the liver against BA toxicity. PMID:25545350

  7. The dynamics of methionine supply and demand during early development.

    PubMed

    McBreairty, Laura E; Bertolo, Robert F

    2016-06-01

    Methionine is an indispensable amino acid that, when not incorporated into protein, is converted into the methyl donor S-adenosylmethionine as entry into the methionine cycle. Following transmethylation, homocysteine is either remethylated to reform methionine or irreversibly trans-sulfurated to form cysteine. Methionine flux to transmethylation and to protein synthesis are both high in the neonate and this review focuses on the dynamics of methionine supply and demand during early development, when growth requires expansion of pools of protein and transmethylation products such as creatine and phosphatidylcholine (PC). The nutrients folate and betaine (derived from choline) donate a methyl group during remethylation, providing an endogenous supply of methionine to meet the methionine demand. During early development, variability in the dietary supply of these methionine cycle-related nutrients can affect both the supply and the demand of methionine. For example, a greater need for creatine synthesis can limit methionine availability for protein and PC synthesis, whereas increased availability of remethylation nutrients can increase protein synthesis if dietary methionine is limiting. Moreover, changes to methyl group availability early in life can lead to permanent changes in epigenetic patterns of DNA methylation, which have been implicated in the early origins of adult disease phenomena. This review aims to summarize how changes in methyl supply and demand can affect the availability of methionine for various functions and highlights the importance of variability in methionine-related nutrients in the infant diet. PMID:27177124

  8. Poly(L-lysine)-g-poly(D,L-lactic-co-glycolic acid) micelles for low cytotoxic biodegradable gene delivery carriers.

    PubMed

    Jeong, Ji Hoon; Park, Tae Gwan

    2002-07-18

    Poly(lactic-co-glycolic acid) (PLGA)-grafted poly(L-lysine) (PLL) (PLL-g-PLGA) was synthesized to demonstrate its micelle-forming property in an aqueous solution. The micelles were used as a gene delivery carrier. The hydrodynamic diameter of PLL-g-PLGA micelles in an aqueous solution was ca. 149 nm with a narrow size distribution. Critical micelle concentration (cmc) was 9.6 mg/l. The PLL-g-PLGA micelles could be used to produce compact nanoparticulate complexes with plasmid DNA, which could efficiently protect the complexed DNA from enzymatic degradation by DNase I. The micelle/DNA complexes had highly compacted structure sized between 200-300 nm with a positive surface charge value. The PLL-g-PLGA micelles exhibited much higher transfection efficiency with lower cytotoxicity than PLL. Here, we demonstrated that biodegradable and cationic PLL-g-PLGA micelles could be used as an effective DNA condensation carrier for gene delivery system.

  9. Functional impact of polar and acidic substitutions in the lactose repressor hydrophobic monomer.monomer interface with a buried lysine.

    PubMed

    Zhan, Hongli; Sun, Zhifei; Matthews, Kathleen Shive

    2009-02-17

    Despite predicted energetic penalties, the charged K84 side chains of tetrameric lactose repressor protein (LacI) are found buried within the highly hydrophobic monomer.monomer interface that includes side chains of V94 and V96. Once inducer binding has occurred, these K84 side chains move to interact with the more solvent-exposed side chains of D88 and E100'. Previous studies demonstrated that hydrophobic substitutions for K84 increased protein stability and significantly impaired the allosteric response. These results indicated that enhanced hydrophobic interactions at the monomer.monomer interface remove the energetic driving force of the buried charges, decreasing the likelihood of a robust conformational change and stabilizing the structure. We hypothesized that creating a salt bridge network with the lysine side chains by including nearby negatively charged residues might result in a similar outcome. To that end, acidic residues, D and E, and their neutral amides, N and Q, were substituted for the valines at positions 94 and 96. These variants exhibited one or more of the following functional changes: weakened inducer binding, impaired allosteric response, and diminished protein stability. For V96D and V96E, ion pair formation with K84 appears optimal, and the loss of inducer response exceeds that of the hydrophobic K84A and -L variants. However, impacts on functional properties indicate that stabilizing the buried positive charge with polar or ion pair interactions is not functionally equivalent to structural stabilization via hydrophobic enhancement. PMID:19166325

  10. Intracellular traffic of the lysine and glutamic acid rich protein KERP1 reveals features of endomembrane organization in Entamoeba histolytica.

    PubMed

    Perdomo, Doranda; Manich, Maria; Syan, Sylvie; Olivo-Marin, Jean-Christophe; Dufour, Alexandre C; Guillén, Nancy

    2016-08-01

    The development of amoebiasis is influenced by the expression of the lysine and glutamic acid rich protein 1 (KERP1), a virulence factor involved in Entamoeba histolytica adherence to human cells. Up to date, it is unknown how the protein transits the parasite cytoplasm towards the plasma membrane, specially because this organism lacks a well-defined endoplasmic reticulum (ER) and Golgi apparatus. In this work we demonstrate that KERP1 is present at the cell surface and in intracellular vesicles which traffic in a pathway that is independent of the ER-Golgi anterograde transport. The intracellular displacement of vesicles enriched in KERP1 relies on the actin-rich cytoskeleton activities. KERP1 is also present in externalized vesicles deposited on the surface of human cells. We further report the interactome of KERP1 with its association to endomembrane components and lipids. The model for KERP1 traffic here proposed hints for the first time elements of the endocytic and exocytic paths of E. histolytica. PMID:26857352

  11. A lysine- and glutamic acid-rich protein, KERP1, from Entamoeba histolytica binds to human enterocytes.

    PubMed

    Seigneur, Marie; Mounier, Joelle; Prevost, Marie-Christine; Guillén, Nancy

    2005-04-01

    Contact-dependent cytolysis of host cells by Entamoeba histolytica is an important hallmark of amoebiasis that points out the importance of molecules involved in the interaction between the parasite and the human cells. To decipher the molecular and cellular mechanisms supporting the invasion of the intestinal epithelium by E. histolytica, we analysed proteins involved in the interaction of the parasite with enterocytes. Affinity chromatography revealed several amoebic proteins interacting with purified brush border of differentiated Caco2 cells. Among them were found the intermediate subunit of the Gal/GalNAc lectin, an alpha-actinin-like protein and two new proteins KERP1 and KERP2 rich in lysine and glutamic acid. In silico analysis revealed the presence of KERP2 in the closely related non-pathogenic amoeba species Entamoeba dispar but not of KERP1. In additon, polymerase chain reaction analysis allowed to suggest the absence of kerp1 homologous gene in E. dispar. Therefore, we concentrated on the cellular analysis of KERP1. Cloning of the KERP1-encoding gene, production of a recombinant protein in Escherichia coli and production of a specific antibody allowed us to show the following properties: (i) purified KERP1 binds to epithelial cell surface, (ii) KERP1 is located on the plasma membrane and in vesicles of trophozoites and (iii) KERP1 is delivered in the interstitial area between the trophozoites and the intestinal cells.

  12. A leptin derived 30-amino-acid peptide modified pegylated poly-L-lysine dendrigraft for brain targeted gene delivery.

    PubMed

    Liu, Yang; Li, Jianfeng; Shao, Kun; Huang, Rongqin; Ye, Liya; Lou, Jinning; Jiang, Chen

    2010-07-01

    The blood-brain barrier is the major obstacle that prevents diagnostic and therapeutic drugs being delivered to the central nervous systems in order to exert their effects. Specific ligand-receptor binding mediated endocytosis is one of the possible strategies to cross this barrier. A 30-amino-acid peptide (leptin30) derived from an endogenic hormone-leptin is exploited as brain-targeting ligand as it is reported to possess the same brain accumulation efficiency after intravenous injection. Dendrigraft poly-L-lysine (DGL) is used as non-viral gene vector in this study. DGL-PEG-Leptin30 was complexed with plasmid DNA yielding nanoparticles (NPs). The cellular uptake characteristic and mechanism were explored in brain capillary endothelial cells (BCECs) which express leptin receptors. Furthermore, brain parenchyma microglia cells such as BV-2 cells expressing leptin receptors could promote ligand-receptor mediated endocytosis leading to enhanced gene transfection ability of DGL-PEG-Leptin30/DNA NPs. The targeted NPs were proved to be transported across in vitro BBB model effectively and accumulate more in brains after i.v. resulting in a relatively high gene transfection efficiency both in vitro and in vivo. Besides, the NPs showed low cytotoxicity after in vitro transfection. Thus, DGL-PEG-Leptin30 provides a safe and noninvasive approach for the delivery of gene across the blood-brain barrier.

  13. Proline-glutamic acid-proline-lysine repetition peptide as an antigen for the serological diagnosis of strangles.

    PubMed

    Hobo, S; Niwa, H; Anzai, T

    2008-04-12

    The reactivity of the proline-glutamic acid-proline-lysine (PEPK) repetition peptide antigen in 3176 serum samples was investigated to evaluate its utility as an antigen for the serological diagnosis of strangles. The reactivity of the sera of horses infected with Streptococcus equi subspecies equi was high when the peptide had several PEPK repetitions. However, as the number of PEPK repetitions increased, the reactivity of the antigen with the sera of horses infected with Streptococcus equi subspecies zooepidemicus also increased. In horses infected experimentally with S equi, the reactivity of the PEPK antigen with five repetitions increased one week after inoculation and continued to increase during the following four weeks. The optical density (OD) values of test sera from horses infected experimentally with S equi and sera from horses that had recovered from strangles were high. The od values of sera from horses that had recovered from an experimental infection with S zooepidemicus and of sera from healthy horses were comparatively low.

  14. Proline-glutamic acid-proline-lysine peptide set as a specific antigen for the serological diagnosis of strangles.

    PubMed

    Hobo, S; Niwa, H; Anzai, T

    2006-11-01

    The reactivity of synthesised peptide sets for the M-like proteins SeM and SzPSe with sera from horses infected with Streptococcus equi or Streptococcus zooepidemicus, or control horses, was investigated by an ELISA. Seventeen horses were infected experimentally with S equi or S zooepidemicus, convalescent sera were obtained from 25 horses and control sera were obtained from 1945 horses. The serum antibody responses of individual horses to the peptide sets were highly variable. Some of the peptide sets for SeM reacted strongly with the sera from the horses infected experimentally with S equi, but also reacted with sera from some of the horses infected experimentally with S zooepidemicus. However, the proline-glutamic acid-proline-lysine (PEPK) repeats peptide set, synthesised from the PEPK repeats areas of SzPSe, reacted most strongly with the sera from the horses infected experimentally with S equi and the horses convalescing from strangles, and reacted only minimally with the sera from the horses infected experimentally with S zooepidemicus and the control horses.

  15. A review of methionine dependency and the role of methionine restriction in cancer growth control and life-span extension.

    PubMed

    Cavuoto, Paul; Fenech, Michael F

    2012-10-01

    Methionine is an essential amino acid with many key roles in mammalian metabolism such as protein synthesis, methylation of DNA and polyamine synthesis. Restriction of methionine may be an important strategy in cancer growth control particularly in cancers that exhibit dependence on methionine for survival and proliferation. Methionine dependence in cancer may be due to one or a combination of deletions, polymorphisms or alterations in expression of genes in the methionine de novo and salvage pathways. Cancer cells with these defects are unable to regenerate methionine via these pathways. Defects in the metabolism of folate may also contribute to the methionine dependence phenotype in cancer. Selective killing of methionine dependent cancer cells in co-culture with normal cells has been demonstrated using culture media deficient in methionine. Several animal studies utilizing a methionine restricted diet have reported inhibition of cancer growth and extension of a healthy life-span. In humans, vegan diets, which can be low in methionine, may prove to be a useful nutritional strategy in cancer growth control. The development of methioninase which depletes circulating levels of methionine may be another useful strategy in limiting cancer growth. The application of nutritional methionine restriction and methioninase in combination with chemotherapeutic regimens is the current focus of clinical studies.

  16. Two amino acid-based superlow fouling polymers: poly(lysine methacrylamide) and poly(ornithine methacrylamide).

    PubMed

    Liu, Qingsheng; Li, Wenchen; Singh, Anuradha; Cheng, Gang; Liu, Lingyun

    2014-07-01

    We developed and investigated two new antifouling zwitterionic polymers, poly(lysine methacrylamide) (pLysAA) and poly(ornithine methacrylamide) (pOrnAA), both derived from natural amino acids - lysine and ornithine, respectively. The pLysAA and pOrnAA brushes were grafted on gold via the surface-initiated photoiniferter-mediated polymerization, with the polymer film thickness controlled by the UV-irradiation time. Nonspecific adsorption from human blood serum and plasma was investigated by surface plasmon resonance. Results show that the adsorption level decreased with the increasing film thickness. With the thin films of ∼14.5 nm, the minimal adsorption on pLysAA was 3.9 ng cm(-2) from serum and 5.4 ng cm(-2) from plasma, whereas the lowest adsorption on pOrnAA was 1.8 and 3.2 ng cm(-2), from serum and plasma, respectively. Such protein resistance is comparable to other widely reported antifouling surfaces such as poly(sulfobetaine methacrylate) and polyacrylamide, with a much thinner polymer film thickness. Both pLysAA and pOrnAA showed better protein resistance than the previously reported serine-based poly(serine methacrylate), whereas the pOrnAA is the best among three. The pLysAA- and pOrnAA-grafted surfaces also highly resisted the endothelial cell attachment and Escherichia coli K12 bacterial adhesion. Nanogels made of pLysAA and pOrnAA were found to be ultrastable in undiluted serum, with no aggregation observed after culturing for 24h. Dextran labeled with fluorescein isothiocyanate (FITC-dextran) was encapsulated in nanogels as a model drug. The encapsulated FITC-dextran exhibited controlled release from the pOrnAA nanogels. The superlow fouling, biomimetic and multifunctional properties of pLysAA and pOrnAA make them promising materials for a wide range of applications, such as implant coating, drug delivery and biosensing. PMID:24613545

  17. Possible Evidence of Amide Bond Formation Between Sinapinic Acid and Lysine-Containing Bacterial Proteins by Matrix-Assisted Laser Desorption/Ionization (MALDI) at 355 nm

    NASA Astrophysics Data System (ADS)

    Fagerquist, Clifton K.; Sultan, Omar; Carter, Michelle Q.

    2012-12-01

    We previously reported the apparent formation of matrix adducts of 3,5-dimethoxy-4-hydroxy-cinnamic acid (sinapinic acid or SA) via covalent attachment to disulfide bond-containing proteins (HdeA, Hde, and YbgS) from bacterial cell lysates ionized by matrix-assisted laser desorption/ionization (MALDI) time-of-flight-time-of-flight tandem mass spectrometry (TOF-TOF-MS/MS) and post-source decay (PSD). We also reported the absence of adduct formation when using α-cyano-4-hydroxycinnamic acid (CHCA) matrix. Further mass spectrometric analysis of disulfide-intact and disulfide-reduced over-expressed HdeA and HdeB proteins from lysates of gene-inserted E. coli plasmids suggests covalent attachment of SA occurs not at cysteine residues but at lysine residues. In this revised hypothesis, the attachment of SA is preceded by formation of a solid phase ammonium carboxylate salt between SA and accessible lysine residues of the protein during sample preparation under acidic conditions. Laser irradiation at 355 nm of the dried sample spot results in equilibrium retrogradation followed by nucleophilic attack by the amine group of lysine at the carbonyl group of SA and subsequent amide bond formation and loss of water. The absence of CHCA adducts suggests that the electron-withdrawing effect of the α-cyano group of this matrix may inhibit salt formation and/or amide bond formation. This revised hypothesis is supported by dissociative loss of SA (-224 Da) and the amide-bound SA (-206 Da) from SA-adducted HdeA and HdeB ions by MS/MS (PSD). It is proposed that cleavage of the amide-bound SA from the lysine side-chain occurs via rearrangement involving a pentacyclic transition state followed by hydrogen abstraction/migration and loss of 3-(4-hydroxy-3,5-dimethoxyphenyl)prop-2-ynal (-206 Da).

  18. Effect of l-lysine-assisted surface grafting for nano-hydroxyapatite on mechanical properties and in vitro bioactivity of poly(lactic acid-co-glycolic acid).

    PubMed

    Liuyun, Jiang; Lixin, Jiang; Chengdong, Xiong; Lijuan, Xu; Ye, Li

    2016-01-01

    It is promising and challenging to study surface modification for nano-hydroxyapatite to improve the dispersion and enhance the mechanical properties and bioactivity of poly(lactic acid-co-glycolic acid). In this paper, we designed an effective new surface grafting with the assist of l-lysine for nano-hydroxyapatite, and the nano-hydroxyapatite surface grafted with the assist of l-lysine (g-nano-hydroxyapatite) was incorporated into poly(lactic acid-co-glycolic acid) to develop a series of g-nano-hydroxyapatite/poly(lactic acid-co-glycolic acid) nano-composites. The surface modification reaction for nano-hydroxyapatite, the mechanical properties, and in vitro human osteoblast-like cell (MG-63) response were characterized and investigated by Fourier transformation infrared, thermal gravimetric analysis, dispersion test, electromechanical universal tester, differential scanning calorimeter measurements, and in vitro cells culture experiment. The results showed that the grafting amount on the surface of nano-hydroxyapatite was enhanced with the increase of l-lysine, and the dispersion of nano-hydroxyapatite was improved more, so that it brought about better promotion crystallization and more excellent mechanical enhancement effect for poly(lactic acid-co-glycolic acid), comparing with the unmodified nano-hydroxyapatite. Moreover, the cells' attachment and proliferation results confirmed that the incorporation of the g-nano-hydroxyapatite into poly(lactic acid-co-glycolic acid) exhibited better biocompatibility than poly(lactic acid-co-glycolic acid). The above results indicated that the new surface grafting with the assist of l-lysine for nano-hydroxyapatite was an ideal novel surface modification method, which brought about better mechanical enhancement effect and in vitro bioactivity for poly(lactic acid-co-glycolic acid) with adding higher g-nano-hydroxyapatite content, suggesting it had a great potential to be used as bone fracture internal fixation materials

  19. Nuclear magnetic resonance studies of amino acids and proteins. Side-chain mobility of methionine in the crystalline amonio acid and in crystallne sperm whale (Physeter catodon) myoglobin

    SciTech Connect

    Keniry, M.A.; Rothgeb, T.M.; Smith, R.L.; Gutowsky, H.S.; Oldfield, E.

    1983-04-12

    Deuterium (/sup 2/H) nuclear magnetic resonance (NMR) spectra and spin-lattice relaxation times (T/sub 1/) were obtained of L-(epsilon-/sup 2/H/sub 3/)methionine, L-(epsilon-/sup 2/H/sub 3/)methionine in a D,L lattice, and (S-methyl-/sup 2/H/sub 3/)methionine in the crystalline solid state, as a function of temperature, in addition to obtaining /sup 2/H T/sub 1/ and line-width results as a function of temperature on (epsilon-/sup 2/H/sub 3/)methionine-labeled sperm whale (Physeter catodon) myoglobins by using the method of magnetic ordering. Also recorded were /sup 13/C cross-polarization ''magic-angle'' sample-spinning NMR spectra of (epsilon-/sup 13/C)methionine-labeled crystalline cyanoferrimyoglobin (at 37.7 MHz, corresponding to a magnetic field strength of 3.52 T) and of the same protein in aqueous solution. (JMT)

  20. Quantum Computational Calculations of the Ionization Energies of Acidic and Basic Amino Acids: Aspartate, Glutamate, Arginine, Lysine, and Histidine

    NASA Astrophysics Data System (ADS)

    de Guzman, C. P.; Andrianarijaona, M.; Lee, Y. S.; Andrianarijaona, V.

    An extensive knowledge of the ionization energies of amino acids can provide vital information on protein sequencing, structure, and function. Acidic and basic amino acids are unique because they have three ionizable groups: the C-terminus, the N-terminus, and the side chain. The effects of multiple ionizable groups can be seen in how Aspartate's ionizable side chain heavily influences its preferred conformation (J Phys Chem A. 2011 April 7; 115(13): 2900-2912). Theoretical and experimental data on the ionization energies of many of these molecules is sparse. Considering each atom of the amino acid as a potential departing site for the electron gives insight on how the three ionizable groups affect the ionization process of the molecule and the dynamic coupling between the vibrational modes. In the following study, we optimized the structure of each acidic and basic amino acid then exported the three dimensional coordinates of the amino acids. We used ORCA to calculate single point energies for a region near the optimized coordinates and systematically went through the x, y, and z coordinates of each atom in the neutral and ionized forms of the amino acid. With the calculations, we were able to graph energy potential curves to better understand the quantum dynamic properties of the amino acids. The authors thank Pacific Union College Student Association for providing funds.

  1. Distinct Paths for Basic Amino Acid Export in Escherichia coli: YbjE (LysO) Mediates Export of l-Lysine

    PubMed Central

    Pathania, Amit

    2015-01-01

    ABSTRACT In Escherichia coli, argO encodes an exporter for l-arginine (Arg) and its toxic analogue canavanine (CAN), and its transcriptional activation and repression, by Arg and l-lysine (Lys), respectively, are mediated by the regulator ArgP. Accordingly argO and argP mutants are CAN supersensitive (CANss). We report the identification of ybjE as a gene encoding a predicted inner membrane protein that mediates export of Lys, and our results confirm the previous identification with a different approach of YbjE as a Lys exporter, reported by Ueda and coworkers (T. Ueda, Y. Nakai, Y. Gunji, R. Takikawa, and Y. Joe, U.S. patents 7,629,142 B2 [December 2009] and 8,383,363 B1 [February 2013] and European patent 1,664,318 B1 [September 2009]). ybjE was isolated as a multicopy suppressor of the CANss phenotype of a strain lacking ArgO. The absence of YbjE did not confer a CANss phenotype but instead conferred hypersensitivity to the lysine antimetabolite thialysine and led to growth inhibition by the dipeptide lysylalanine, which is associated with elevated cellular Lys content. YbjE overproduction resulted in Lys excretion and syntrophic cross-feeding of a Lys auxotroph. Constitutive overexpression of argO promoted Lys cross-feeding that is indicative of a latent Lys export potential of ArgO. Arg modestly repressed ybjE transcription in an ArgR-dependent manner, and ArgR displayed Arg-sensitive binding to the ybjE promoter region in vitro. Our studies suggest that the reciprocal repression of argO and ybjE, respectively, by Lys and Arg confers the specificity for basic amino acid export by distinct paths and that such cross-repression contributes to maintenance of cytoplasmic Arg/Lys balance. We propose that YbjE be redesignated LysO. IMPORTANCE This work ascribes a lysine export function to the product of the ybjE gene of Escherichia coli, leading to a physiological scenario wherein two proteins, ArgO and YbjE, perform the task of separately exporting arginine and

  2. The catabolic enzyme methionine gamma-lyase limits methionine accumulation in potato tubers.

    PubMed

    Huang, Tengfang; Joshi, Vijay; Jander, Georg

    2014-09-01

    Increasing methionine in potato tubers is desirable, both to increase the availability of this limiting essential amino acid and to enhance the aroma of baked and fried potatoes. Previous attempts to elevate potato methionine content using transgenic approaches have focused on increasing methionine biosynthesis. Higher isoleucine accumulation in these transgenic tubers suggested that the potatoes compensate for increased methionine biosynthesis with enhanced catabolism via methionine gamma-lyase (MGL), thereby producing 2-ketybutyrate for isoleucine biosynthesis. In the current study, we show that potato StMGL1 encodes a functional MGL in potato tubers. In planta silencing of StMGL1 results in an increased methionine to isoleucine ratio in the free amino acid profile of potato tubers and, in some transgenic lines, elevated accumulation of free methionine. In both wild-type and transgenic tubers, the ratio of methionine to isoleucine is negatively correlated with the level of StMGL1 transcript. A three-dimensional distribution of free amino acids in potato tubers is also described.

  3. 21 CFR 172.372 - N-Acetyl-L-methionine.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... amino acid methionine formed by addition of an acetyl group to the alpha-amino group of methionine. It... amino acid) by weight of the total protein of the finished food, including the amount naturally present... of the additive contained therein. (2) The amounts of additive and each amino acid contained in...

  4. 21 CFR 172.372 - N-Acetyl-L-methionine.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... Abstracts Service Registry No. 65-82-7) is the derivative of the amino acid methionine formed by addition of... percent L- and DL-methionine (expressed as the free amino acid) by weight of the total protein of the...) The amounts of additive and each amino acid contained in any mixture. (3) Adequate directions for...

  5. 21 CFR 172.372 - N-Acetyl-L-methionine.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... amino acid methionine formed by addition of an acetyl group to the alpha-amino group of methionine. It... amino acid) by weight of the total protein of the finished food, including the amount naturally present... of the additive contained therein. (2) The amounts of additive and each amino acid contained in...

  6. 21 CFR 172.372 - N-Acetyl-L-methionine.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... amino acid methionine formed by addition of an acetyl group to the alpha-amino group of methionine. It... amino acid) by weight of the total protein of the finished food, including the amount naturally present... of the additive contained therein. (2) The amounts of additive and each amino acid contained in...

  7. The regulatory effect of citric acid on the co-production of poly(ε-lysine) and poly(L-diaminopropionic acid) in Streptomyces albulus PD-1.

    PubMed

    Xia, Jun; Xu, Zhaoxian; Xu, Hong; Feng, Xiaohai; Bo, Fangfang

    2014-10-01

    Streptomyces albulus PD-1 can co-produce antimicrobial homo-polymers poly(ε-lysine) (ε-PL) and poly(L-diaminopropionic acid) (PDAP). In this study, a novel feeding strategy of citric acid coupled with glucose-(NH4)2SO4 feeding was employed to S. albulus PD-1. When the pH of the culture broth dropped to 4.0, the feeding solution was added continuously to maintain the concentrations of glucose and citric acid at 10 and 4 g L(-1), respectively. As a result, the final concentration of ε-PL increased from 21.7 to 29.7 g L(-1) and the final concentration of PDAP decreased from 4.8 to 3.2 g L(-1). Assays on intracellular nucleotide levels and key enzyme activities were performed to elucidate the underlying regulation mechanism. The addition of citric acid increased NADH/NAD(+) ratio and decreased intracellular ATP level; meanwhile, the activities of pyruvate kinase, citrate synthase and isocitrate dehydrogenase decreased while aspartate aminotransferase activity increased. Therefore, we deduced that citric acid feeding resulted in metabolic flux redistribution at the node of phosphoenolpyruvate; the metabolic pathway from phosphoenolpyruvate directed into tricarboxylic acid cycle was weakened and thus PDAP production was inhibited. On the other hand, the metabolic pathway from phosphoenolpyruvate directed into oxaloacetate and L-aspartate was enhanced, thereby improving ε-PL production. This fermentation strategy may be potentially useful in ε-PL production because it can effectively inhibit the formation of by-products, such as PDAP.

  8. The regulatory effect of citric acid on the co-production of poly(ε-lysine) and poly(L-diaminopropionic acid) in Streptomyces albulus PD-1.

    PubMed

    Xia, Jun; Xu, Zhaoxian; Xu, Hong; Feng, Xiaohai; Bo, Fangfang

    2014-10-01

    Streptomyces albulus PD-1 can co-produce antimicrobial homo-polymers poly(ε-lysine) (ε-PL) and poly(L-diaminopropionic acid) (PDAP). In this study, a novel feeding strategy of citric acid coupled with glucose-(NH4)2SO4 feeding was employed to S. albulus PD-1. When the pH of the culture broth dropped to 4.0, the feeding solution was added continuously to maintain the concentrations of glucose and citric acid at 10 and 4 g L(-1), respectively. As a result, the final concentration of ε-PL increased from 21.7 to 29.7 g L(-1) and the final concentration of PDAP decreased from 4.8 to 3.2 g L(-1). Assays on intracellular nucleotide levels and key enzyme activities were performed to elucidate the underlying regulation mechanism. The addition of citric acid increased NADH/NAD(+) ratio and decreased intracellular ATP level; meanwhile, the activities of pyruvate kinase, citrate synthase and isocitrate dehydrogenase decreased while aspartate aminotransferase activity increased. Therefore, we deduced that citric acid feeding resulted in metabolic flux redistribution at the node of phosphoenolpyruvate; the metabolic pathway from phosphoenolpyruvate directed into tricarboxylic acid cycle was weakened and thus PDAP production was inhibited. On the other hand, the metabolic pathway from phosphoenolpyruvate directed into oxaloacetate and L-aspartate was enhanced, thereby improving ε-PL production. This fermentation strategy may be potentially useful in ε-PL production because it can effectively inhibit the formation of by-products, such as PDAP. PMID:24752482

  9. Efficacy of a Complex of 5-Aminolevulinic Acid and Glycyl-Histidyl-Lysine Peptide on Hair Growth

    PubMed Central

    Sim, Hyun Bo; Jang, Yong Hyun; Lee, Seok-Jong; Kim, Do Won; Yim, Soon-Ho

    2016-01-01

    Background Pattern hair loss is a very common problem. Although effective therapeutics for the treatment of pattern hair loss have been used, novel therapeutic modalities are still required to enhance hair growth. Objective We investigated the efficacy and safety of a complex (ALAVAX) of 5-aminolevulinic acid (5-ALA) and glycyl-histidyl-lysine (GHK) peptide for the treatment of pattern hair loss. Methods Forty-five patients with male pattern hair loss were treated with ALAVAX 100 mg/ml (group A), ALAVAX 50 mg/ml (group B) or placebo (group C) once a day for 6 months. Total hair count, hair length, hair thickness, patient's assessment and adverse events were evaluated at month 1, 3, and 6. Results An increase in hair count for 6 months was 52.6 (p<0.05) in group A, 71.5 (p<0.05) in group B, and 9.6 in group C. The ratio of changes in hair count between group B (2.38) and group C (1.21) at 6 months showed a statistically significant difference (p<0.05). The proportion above good satisfaction was higher in group A (26.7%) than in the other groups (group B: 14.3%, group C: 7.1%). There was no statistically significant difference in hair length and hair thickness among 3 groups at 6 months. There was no adverse event in 3 groups. Conclusion Our study showed that a complex of 5-ALA and GHK peptide may be considered as one of the complementary agents for the treatment of male pattern hair loss. PMID:27489425

  10. The catabolic function of the alpha-aminoadipic acid pathway in plants is associated with unidirectional activity of lysine-oxoglutarate reductase, but not saccharopine dehydrogenase.

    PubMed Central

    Zhu, X; Tang, G; Galili, G

    2000-01-01

    Whereas plants and animals use the alpha-aminoadipic acid pathway to catabolize lysine, yeast and fungi use the very same pathway to synthesize lysine. These two groups of organisms also possess structurally distinct forms of two enzymes in this pathway, namely lysine-oxoglutarate reductase (lysine-ketoglutarate reductase; LKR) and saccharopine dehydrogenase (SDH): in plants and animals these enzymes are linked on to a single bifunctional polypeptide, while in yeast and fungi they exist as separate entities. In addition, yeast LKR and SDH possess bi-directional activities, and their anabolic function is regulated by complex transcriptional and post-transcriptional controls, which apparently ascertain differential accumulation of intermediate metabolites; in plants, the regulation of the catabolic function of these two enzymes is not known. To elucidate the regulation of the catabolic function of plant bifunctional LKR/SDH enzymes, we have used yeast as an expression system to test whether a plant LKR/SDH also possesses bi-directional LKR and SDH activities, similar to the yeast enzymes. The Arabidopsis enzyme complemented a yeast SDH, but not LKR, null mutant. Identical results were obtained when deletion mutants encoding only the LKR or SDH domains of this bifunctional polypeptide were expressed individually in the yeast cells. Moreover, activity assays showed that the Arabidopsis LKR possessed catabolic, but not anabolic, activity, and its uni-directional activity stems from its structure rather than its linkage to SDH. Our results suggest that the uni-directional activity of LKR plays an important role in regulating the catabolic function of the alpha-amino adipic acid pathway in plants. PMID:10998364

  11. L-leucine, L-methionine, and L-phenylalanine share a Na(+)/K (+)-dependent amino acid transporter in shrimp hepatopancreas.

    PubMed

    Duka, Ada; Ahearn, Gregory A

    2013-08-01

    Hepatopancreatic brush border membrane vesicles (BBMV), made from Atlantic White shrimp (Litopenaeus setiferus), were used to characterize the transport properties of (3)H-L-leucine influx by these membrane systems and how other essential amino acids and the cations, sodium and potassium, interact with this transport system. (3)H-L-leucine uptake by BBMV was pH-sensitive and occurred against transient transmembrane concentration gradients in both Na(+)- and K(+)-containing incubation media, suggesting that either cation was capable of providing a driving force for amino acid accumulation. (3)H-L-leucine uptake in NaCl or KCl media were each three times greater in acidic pH (pH 5.5) than in alkaline pH (pH 8.5). The essential amino acid, L-methionine, at 20 mM significantly (p < 0.0001) inhibited the 2-min uptakes of 1 mM (3)H-L-leucine in both Na(+)- and K(+)-containing incubation media. The residual (3)H-L-leucine uptake in the two media were significantly greater than zero (p < 0.001), but not significantly different from each other (p > 0.05) and may represent an L-methionine- and cation-independent transport system. (3)H-L-leucine influxes in both NaCl and KCl incubation media were hyperbolic functions of [L-leucine], following the carrier-mediated Michaelis-Menten equation. In NaCl, (3)H-L-leucine influx displayed a low apparent K M (high affinity) and low apparent J max, while in KCl the transport exhibited a high apparent K M (low affinity) and high apparent J max. L-methionine or L-phenylalanine (7 and 20 mM) were competitive inhibitors of (3)H-L-leucine influxes in both NaCl and KCl media, producing a significant (p < 0.01) increase in (3)H-L-leucine influx K M, but no significant response in (3)H-L-leucine influx J max. Potassium was a competitive inhibitor of sodium co-transport with (3)H-L-leucine, significantly (p < 0.01) increasing (3)H-L-leucine influx K M in the presence of sodium, but having negligible effect on (3)H-L-leucine influx J

  12. Methionine metabolism in Yucatan miniature swine.

    PubMed

    McBreairty, Laura E

    2016-06-01

    Methionine is an essential amino acid which when not incorporated into protein, can be converted to S-adenosylmethionine, the universal methyl donor in over 200 transmethylation reactions, which include creatine and phosphatidylcholine (PC) synthesis, as well as deoxyribonucleic acid (DNA) methylation. Following transmethylation, homocysteine is formed, which can be converted to cysteine via transsulfuration or remethylated to methionine by receiving a methyl group from folate or betaine. Changes to methyl group availability in utero can lead to permanent changes in epigenetic patterns of DNA methylation, which has been implicated in "fetal programming", a phenomenon associated with poor nutrition during fetal development that results in low birth weight and disease in later life. It has been shown that programming can also occur in the neonate. Our global objective was to understand how the variability of nutrients involved in methionine metabolism can affect methionine and methyl group availability. We hypothesize that nutrients that converge on methionine metabolism can affect methionine availability for its various functions. In this thesis, we used intrauterine growth restricted (IUGR) piglets to investigate whether a global nutritional insult in utero can lead to a perturbed methionine metabolism. Our results demonstrate that IUGR piglets have a lower capacity to dispose of homocysteine via both transsulfuration and remethylation pathways, as well as a lower incorporation of methyl groups into PC. The second objective of this thesis was to determine whether variation in methionine supply and demand can affect methionine availability. We demonstrated that stimulating either acute or chronic creatine synthesis leads to lower methyl incorporation into protein and PC in pigs. Furthermore, when methionine is limiting, supplementation with either folate or betaine leads to higher methionine availability for protein synthesis. Finally, because creatine is

  13. Conserved methionines in chloroplasts.

    PubMed

    Sundby, Cecilia; Härndahl, Ulrika; Gustavsson, Niklas; Ahrman, Emma; Murphy, Denis J

    2005-01-17

    Heat shock proteins counteract heat and oxidative stress. In chloroplasts, a small heat shock protein (Hsp21) contains a set of conserved methionines, which date back to early in the emergence of terrestrial plants. Methionines M49, M52, M55, M59, M62, M67 are located on one side of an amphipathic helix, which may fold back over two other conserved methionines (M97 and M101), to form a binding groove lined with methionines, for sequence-independent recognition of peptides with an overall hydrophobic character. The sHsps protect other proteins from aggregation by binding to their hydrophobic surfaces, which become exposed under stress. Data are presented showing that keeping the conserved methionines in Hsp21 in a reduced form is a prerequisite to maintain such binding. The chloroplast generates reactive oxygen species under both stress and unstressed conditions, but this organelle is also a highly reducing cellular compartment. Chloroplasts contain a specialized isoform of the enzyme, peptide methionine sulfoxide reductase, the expression of which is light-induced. Recombinant proteins were used to measure that this reductase can restore Hsp21 methionines after sulfoxidation. This paper also describes how methionine sulfoxidation-reduction can be directly assessed by mass spectrometry, how methionine-to-leucine substitution affects Hsp21, and discusses the possible role for an Hsp21 methionine sulfoxidation-reduction cycle in quenching reactive oxygen species. PMID:15680227

  14. Factors influencing methionine toxicity in young bobwhite quail

    USGS Publications Warehouse

    Serafin, J.A.

    1981-01-01

    Young Bobwhite quail (Colinus virginianus) were fed low and adequate protein purified diets with and without excess methionine to evaluate factors affecting methionine toxicity. Growth of quail fed an adequate protein (27%) diet, without supplemental glycine, was depressed by 1.75% and 2.25% excess methionine. Supplemental glycine (.3%) alleviated growth depression caused by 2.25% excess methionine. Quail fed 1.75% and 2.25% excess methionine developed signs of toxicity characterized by weakness, a lowered, outstretched neck when moving, and ataxia. In addition, quail would fall on their sides when disturbed and spin with their heads retracted. These conditions were transient in nature. Growth of quail fed a low protein (18.9%) diet was depressed by 1% and 1.5% excess methionine and DL-homocystine. Quail fed 1% and 1.5% excess methionine in this diet also developed signs of toxicity, the incidence of which was greater and the duration longer than occurred with quail fed adequate protein. Supplementing a low protein (20.15%) diet with .3% or .6% glycine or threonine or a combination of these amino acids did not alleviate growth depression caused by 1.5% excess methionine; however, 2% and 3% supplemental glycine were somewhat effective. Supplements of glycine (2%, 3%) and threonine (1%) completely reversed growth depression from 1% excess methionine but did not influence growth of controls, indicating that both amino acids counteract methionine toxicity. Both glycine and threonine alone improved growth by about the same extent in diets with 1% or 1.5% excess methionine; however, these amino acids alleviated less than 30% of the growth depression resulting from 1.5% excess methionine. The effectiveness of glycine in alleviating methionine toxicity in a low protein diet was decreased, and hemoglobin levels were depressed with 1.5% excess methionine compared to less amounts.

  15. Availability to lactating dairy cows of methionine added to soy lecithins and mixed with a mechanically extracted soybean meal.

    PubMed

    Brake, D W; Titgemeyer, E C; Brouk, M J; Macgregor, C A; Smith, J F; Bradford, B J

    2013-05-01

    We evaluated a product containing methionine mixed with soy lecithins and added to a mechanically extracted soybean meal (meSBM-Met). Lactational responses of cows, plasma methionine concentrations, and in vitro degradation of methionine were measured. Twenty-five Holstein cows were used in a replicated 5 × 5 Latin square design and fed a diet designed to be deficient in methionine or the same diet supplemented either with 4.2 or 8.3g/d of supplemental methionine from a ruminally protected source or with 2.7 or 5.3g/d of supplemental methionine from meSBM-Met. All diets were formulated to provide adequate amounts of metabolizable lysine. Concentration of milk true protein was greater when methionine was provided by the ruminally protected methionine than by meSBM-Met, but milk protein yield was not affected by treatment. Milk yields and concentrations and yields of fat, lactose, solids-not-fat, and milk urea nitrogen were not affected by supplemental methionine. Body condition scores increased linearly when methionine from meSBM-Met was supplemented, but responses were quadratic when methionine was provided from a ruminally protected source. Nitrogen retention was not affected by supplemental methionine. Plasma methionine increased linearly when methionine was supplemented from a ruminally protected source, but plasma methionine concentrations did not differ from the control when supplemental methionine from meSBM-Met was provided. In vitro degradation of supplemental methionine from meSBM-Met was complete within 3h. Data suggest that meSBM-Met provides negligible amounts of metabolizable methionine to dairy cows, and this is likely related to extensive ruminal destruction of methionine; however, cow body condition may be improved by ruminally available methionine provided by meSBM-Met.

  16. QSAR study of anthranilic acid sulfonamides as inhibitors of methionine aminopeptidase-2 using LS-SVM and GRNN based on principal components.

    PubMed

    Shahlaei, Mohsen; Sabet, Razieh; Ziari, Maryam Bahman; Moeinifard, Behzad; Fassihi, Afshin; Karbakhsh, Reza

    2010-10-01

    Quantitative relationships between molecular structure and methionine aminopeptidase-2 inhibitory activity of a series of cytotoxic anthranilic acid sulfonamide derivatives were discovered. We have demonstrated the detailed application of two efficient nonlinear methods for evaluation of quantitative structure-activity relationships of the studied compounds. Components produced by principal component analysis as input of developed nonlinear models were used. The performance of the developed models namely PC-GRNN and PC-LS-SVM were tested by several validation methods. The resulted PC-LS-SVM model had a high statistical quality (R(2)=0.91 and R(CV)(2)=0.81) for predicting the cytotoxic activity of the compounds. Comparison between predictability of PC-GRNN and PC-LS-SVM indicates that later method has higher ability to predict the activity of the studied molecules.

  17. Methionine, pyridoxine and endothelial lesion in rats.

    PubMed

    Hladovec, J

    1980-01-01

    Methionine administered orally to rats produced a prolonged dose-dependent increase in endothelemia. The increase was observed after doses exceeding 100 mg/kg and was inhibited by a simultaneous administration of pyridoxine. The effect of methionine was also inhibited by trihydroxyethylrutoside and acetylsalicyclic acid. Endothelemia was increased furthermore by oral administration of cysteine and cystine and this increase was again inhibited by pyridoxine.

  18. Methionine restriction and lifespan control

    PubMed Central

    Lee, Byung Cheon; Kaya, Alaattin; Gladyshev, Vadim N.

    2016-01-01

    Dietary restriction (DR) without malnutrition is associated with longevity in various organisms. However, it has also been shown that reduced calorie intake is often ineffective in extending lifespan. Selecting optimal dietary regimens for DR studies is complicated, as the same regimen may lead to different outcomes depending on genotype and environmental factors. Recent studies suggested that interventions such as moderate protein restriction with/without adequate nutrition (e.g. particular amino acids or carbohydrates) may have additional beneficial effects mediated by certain metabolic and hormonal factors implicated in the biology of aging, regardless of total calorie intake. In particular, it was shown that restriction of a single amino acid, methionine, can mimic the effects of DR and extend lifespan in various model organisms. We discuss beneficial effects of methionine-restricted (MR) diet, the molecular pathways involved, and the use of this regimen in longevity interventions. PMID:26663138

  19. Proteomic and Biochemical Studies of Lysine Malonylation Suggest Its Malonic Aciduria-associated Regulatory Role in Mitochondrial Function and Fatty Acid Oxidation.

    PubMed

    Colak, Gozde; Pougovkina, Olga; Dai, Lunzhi; Tan, Minjia; Te Brinke, Heleen; Huang, He; Cheng, Zhongyi; Park, Jeongsoon; Wan, Xuelian; Liu, Xiaojing; Yue, Wyatt W; Wanders, Ronald J A; Locasale, Jason W; Lombard, David B; de Boer, Vincent C J; Zhao, Yingming

    2015-11-01

    The protein substrates of sirtuin 5-regulated lysine malonylation (Kmal) remain unknown, hindering its functional analysis. In this study, we carried out proteomic screening, which identified 4042 Kmal sites on 1426 proteins in mouse liver and 4943 Kmal sites on 1822 proteins in human fibroblasts. Increased malonyl-CoA levels in malonyl-CoA decarboxylase (MCD)-deficient cells induces Kmal levels in substrate proteins. We identified 461 Kmal sites showing more than a 2-fold increase in response to MCD deficiency as well as 1452 Kmal sites detected only in MCD-/- fibroblast but not MCD+/+ cells, suggesting a pathogenic role of Kmal in MCD deficiency. Cells with increased lysine malonylation displayed impaired mitochondrial function and fatty acid oxidation, suggesting that lysine malonylation plays a role in pathophysiology of malonic aciduria. Our study establishes an association between Kmal and a genetic disease and offers a rich resource for elucidating the contribution of the Kmal pathway and malonyl-CoA to cellular physiology and human diseases. PMID:26320211

  20. Linkages in thermal copolymers of lysine

    NASA Technical Reports Server (NTRS)

    Fox, S. W.; Suzuki, F.

    1975-01-01

    The thermal copolymerization of lysine with other alpha-amino acids was studied. The identity of the second amino acid influences various properties of the polymer obtained, including the proportion of alpha and epsilon linkages of lysine. A review of linkages in proteinoids indicates alpha and beta linkages for aspartic acid, alpha and gamma linkages for glutamic acid, alpha and epsilon linkages for lysine, and alpha linkages for other amino acids. Thermal proteinoids are thus more complex in types of linkage than are proteins.

  1. Current topics in the biotechnological production of essential amino acids, functional amino acids, and dipeptides.

    PubMed

    Mitsuhashi, Satoshi

    2014-04-01

    Amino acids play important roles in both human and animal nutrition and in the maintenance of health. Here, amino acids are classified into three groups: first, essential amino acids, which are essential to nutrition; second, functional amino acids, recently found to be important in the promotion of physiological functions; and third, dipeptides, which are used to resolve problematic features of specific free amino acids, such as their instability or insolubility. This review focusses on recent researches concerning the microbial production of essential amino acids (lysine and methionine), functional amino acids (histidine and ornithine), and a dipeptide (L-alanyl-L-glutamine). PMID:24679256

  2. The same substitution, glutamic acid----lysine at position 501, occurs in three alloalbumins of Asiatic origin: albumins Vancouver, Birmingham, and Adana.

    PubMed Central

    Huss, K; Madison, J; Ishioka, N; Takahashi, N; Arai, K; Putnam, F W

    1988-01-01

    A strategy is described for identifying structural changes in genetic variants of human serum albumin (alloalbumins). By use of this strategy we have determined an amino acid substitution in three alloalbumins of Asiatic origin. The same amino acid exchange, glutamic acid----lysine at position 501, occurs in albumins Vancouver and Birmingham, both from families that migrated from northern India, and also in albumin Adana from Turkey. This exchange corresponds to a single base mutation in the codon GAG to AAG and accords with the slow mobility of the three albumins at pH 8.6. Each of the three alloalbumins had been reported to be a new variant, yet they have the same substitution. These results emphasize the need for structural study of genetic variants that have been differentiated only by nonspecific physical criteria such as dye binding and electrophoretic mobility. We know of no other description of the substitution involved in an alloalbumin originating from the Indian subcontinent. However, the same change of glutamic acid----lysine at position 501 may be present in several other named variants reported for populations in north India and the surrounding regions. Images PMID:2901102

  3. Effects of Chromium Methionine Supplementation on Blood Metabolites and Fatty Acid Profile of Beef during Late Fattening Period in Holstein Steers

    PubMed Central

    Nejad, Jalil Ghassemi; Lee, Bae-Hun; Kim, Byong-Wan; Ohh, Sang-Jip; Sung, Kyung Il

    2016-01-01

    The objective of this study was to determine the effects of chromium methionine (Cr-Met) chelate supplementation on blood metabolites and fatty acid profile of beef from Holstein steers during late fattening period. Fifteen Holstein steers were allotted randomly into two groups including the control (non Cr-Met feeding, NCM, ave. body weight [BW] = 483±25.7 kg) and the treatment (Cr-Met feeding for 4 months, 4CM, ave. BW = 486±27.5 kg) group. The feeding amount of Cr-Met to animals was limited to 400 ppb/cow/d and was supplemented to total mixed ration. No difference in blood albumin, alkaline phosphatase, urea-nitrogen, calcium, creatine, glucose, total protein, triglyceride, and cholesterol were observed between the treatment groups (p>0.05). The level of high density lipoprotein was higher in the 4CM group than the NCM group, whereas low density lipoprotein was lower in the 4CM group (p<0.05). The fatty acid composition (caprate, laurate, myristate, pentadecanoate, palmitate, palmitoleate, margarate, cis-11 heptadodecanoate, stearate, oleate, trans-vaccenate, linoleate, cis-11 eicosenoate, docosa hexaenoic acid, and docosa pentaenoic acid) of the beef showed no difference between the two groups (p>0.05). The arachidonic acid level tended to be higher in the 4CM than the NCM group (p = 0.07). Cr-Met had no influence (p>0.05) on the ratio of saturated, unsaturated, unsaturated/saturated, monounsaturated/saturated and polyunsaturated/saturated fatty acids whereas the ratio of polyunsaturated fatty acids (PUFA) in the 4CM group was comparatively higher than the NCM group (p<0.05). This study concluded that feeding Cr-Met supplementation in 400 ppb/d to Holstein steers for 4 months during late fattening period can improve some blood metabolites and beef quality by increasing PUFA and gamma-linoleate compositions of beef. PMID:26950869

  4. A molecular dynamics and quantum mechanics/molecular mechanics study of the catalytic reductase mechanism of methionine sulfoxide reductase A: formation and reduction of a sulfenic acid.

    PubMed

    Dokainish, Hisham M; Gauld, James W

    2013-03-12

    The catalytic mechanism of MsrA in Mycobacterium tuberculosis, in which S-methionine sulfoxide (Met-O) is reduced to methionine (Met), has been investigated using docking, molecular dynamics (MD) simulations, and ONIOM (quantum mechanics/molecular mechanics) methods. In addition, the roles of specific active site residues, including an aspartyl (Asp87) near the recycling cysteine, tyrosyls (Tyr44 and Tyr92), and glutamyl (Glu52), have been examined, as well as the general effects of the protein and active site on the nature and properties of mechanistic intermediates. The mechanism is initiated by the transfer of a proton from the catalytic cysteine's thiol (Cys13SH) via a bridging water to the R group carboxylate of Glu52. The now anionic sulfur of Cys13 nucleophilically attacks the substrate's sulfur with concomitant transfer of a proton from Glu52 to the sulfoxide oxygen, generating a sulfurane. The active site enhances the proton affinity of the sulfurane oxygen, which can readily accept a proton from the phenolic hydroxyls of Tyr44 or Tyr92 to give a sulfonium cation. Subsequently, Asp87 and the recycling cysteine (Cys154) can facilitate nucleophilic attack of a solvent water at the Cys13S center of the sulfonium to give a sulfenic acid (Cys13SOH) and Met. For the subsequent reduction of Cys13SOH with intramolecular disulfide bond formation, Asp87 can help facilitate nucleophilic attack of Cys154S at the sulfur of Cys13SOH by deprotonating its thiol. This reduction is found likely to occur readily upon suitable positioning of the active site hydrogen bond network and the sulfur centers of both Cys13 and Cys154. The calculated rate-limiting barrier is in good agreement with experiment.

  5. Characterization of isolated yeast growth response to methionine analogs.

    PubMed

    Saengkerdsub, Suwat; Lingbeck, Jody M; Wilkinson, Heather H; O'Bryan, Corliss A; Crandall, Philip G; Muthaiyan, Arunachalam; Biswas, Debabrata; Ricke, Steven C

    2013-01-01

    Methionine is one of the first limiting amino acids in poultry nutrition. The use of methionine-rich natural feed ingredients, such as soybean meal or rapeseed meal may lead to negative environmental consequences. Amino acid supplementation leads to reduced use of protein-rich ingredients. The objectives of this study were isolation of potentially high content methionine-containing yeasts, quantification of methionine content in yeasts and their respective growth response to methionine analogs. Minimal medium was used as the selection medium and the isolation medium of methionine-producing yeasts from yeast collection and environmental samples, respectively. Two yeasts previously collected along with six additional strains isolated from Caucasian kefir grains, air-trapped, cantaloupe, and three soil samples could grow on minimal medium. Only two of the newly isolated strains, K1 and C1, grew in minimal medium supplied with either methionine analogs ethionine or norleucine at 0.5% (w/v). Based on large subunit rRNA sequences, these isolated strains were identified as Pichia udriavzevii/Issatchenkia orientalis. P. kudriavzevii/I. orentalis is a generally recognized as a safe organism. In addition, methionine produced by K1 and C1 yeast hydrolysate yielded 1.3 ± 0.01 and 1.1 ± 0.01 mg g(-1) dry cell. Yeast strain K1 may be suitable as a potential source of methionine for dietary supplements in organic poultry feed but may require growth conditions to further increase their methionine content. PMID:24007489

  6. Metabolism of 5-methylthioribose to methionine

    SciTech Connect

    Miyazaki, J.H.; Yang, S.F.

    1987-06-01

    During ethylene biosynthesis, the H/sub 3/CS-group of S-adenosylmethionine is released as 5'-methylthioadenosine, which is recycled to methionine via 5-methylthioribose (MTR). In mungbean hypocotyls and cell-free extracts of avocado, (/sup 14/C)MTR was converted into labeled methionine via 2-keto-4-methylthiobutyric acid (KMB) and 2-hydroxy-4-methylthiobutyric acid (HMB), as intermediates. Incubation of (ribose-U-/sup 14/C)MTR with avocado extract resulted in the production of (/sup 14/C)formate, indicating the conversion of MTR to KMB involves a loss of formate, presumably from C-1 of MTR. Tracer studies showed that KMB was converted readily in vivo and in vitro to methionine, while HMB was converted much more slowly. The conversion of KMB to methionine by dialyzed avocado extract requires an amino donor. Among several potential donors examined, L-glutamine was the most efficient. Anaerobiosis inhibited only partially the oxidation of MTR to formate, KMB/HMB, and methionine by avocado extract. The role of O/sub 2/ in the conversion of MTR to methionine is discussed.

  7. Lysine Residues Are Not Required for Proteasome-Mediated Proteolysis of the Auxin/Indole Acidic Acid Protein IAA1.

    PubMed

    Gilkerson, Jonathan; Kelley, Dior R; Tam, Raymond; Estelle, Mark; Callis, Judy

    2015-06-01

    Although many ubiquitin-proteasome substrates have been characterized in plants, very little is known about the corresponding ubiquitin attachment(s) underlying regulated proteolysis. Current dogma asserts that ubiquitin is typically covalently attached to a substrate through an isopeptide bond between the ubiquitin carboxy terminus and a substrate lysyl amino group. However, nonlysine (non-Lys) ubiquitin attachment has been observed in other eukaryotes, including the N terminus, cysteine, and serine/threonine modification. Here, we investigate site(s) of ubiquitin attachment on indole-3-acetic acid1 (IAA1), a short-lived Arabidopsis (Arabidopsis thaliana) Auxin/indole-3-acetic acid (Aux/IAA) family member. Most Aux/IAA proteins function as negative regulators of auxin responses and are targeted for degradation after ubiquitination by the ubiquitin ligase SCF(TIR1/AFB) (for S-Phase Kinase-Associated Protein1, Cullin, F-box [SCF] with Transport Inhibitor Response1 [TIR1]/Auxin Signaling F-box [AFB]) by an interaction directly facilitated by auxin. Surprisingly, using a Histidine-Hemaglutinin (HIS(6x)-HA(3x)) epitope-tagged version expressed in vivo, Lys-less IAA1 was ubiquitinated and rapidly degraded in vivo. Lys-substituted versions of IAA1 localized to the nucleus as Yellow Fluorescent Protein fusions and interacted with both TIR1 and IAA7 in yeast (Saccharomyces cerevisiae) two-hybrid experiments, indicating that these proteins were functional. Ubiquitination on both HIS(6x)-HA(3x)-IAA1 and Lys-less HIS(6x)-HA(3x)-IAA1 proteins was sensitive to sodium hydroxide treatment, indicative of ubiquitin oxyester formation on serine or threonine residues. Additionally, base-resistant forms of ubiquitinated IAA1 were observed for HIS(6x)-HA(3x)-IAA1, suggesting additional lysyl-linked ubiquitin on this protein. Characterization of other Aux/IAA proteins showed that they have diverse degradation rates, adding additional complexity to auxin signaling. Altogether, these data

  8. Lysine Residues Are Not Required for Proteasome-Mediated Proteolysis of the Auxin/Indole Acidic Acid Protein IAA1.

    PubMed

    Gilkerson, Jonathan; Kelley, Dior R; Tam, Raymond; Estelle, Mark; Callis, Judy

    2015-06-01

    Although many ubiquitin-proteasome substrates have been characterized in plants, very little is known about the corresponding ubiquitin attachment(s) underlying regulated proteolysis. Current dogma asserts that ubiquitin is typically covalently attached to a substrate through an isopeptide bond between the ubiquitin carboxy terminus and a substrate lysyl amino group. However, nonlysine (non-Lys) ubiquitin attachment has been observed in other eukaryotes, including the N terminus, cysteine, and serine/threonine modification. Here, we investigate site(s) of ubiquitin attachment on indole-3-acetic acid1 (IAA1), a short-lived Arabidopsis (Arabidopsis thaliana) Auxin/indole-3-acetic acid (Aux/IAA) family member. Most Aux/IAA proteins function as negative regulators of auxin responses and are targeted for degradation after ubiquitination by the ubiquitin ligase SCF(TIR1/AFB) (for S-Phase Kinase-Associated Protein1, Cullin, F-box [SCF] with Transport Inhibitor Response1 [TIR1]/Auxin Signaling F-box [AFB]) by an interaction directly facilitated by auxin. Surprisingly, using a Histidine-Hemaglutinin (HIS(6x)-HA(3x)) epitope-tagged version expressed in vivo, Lys-less IAA1 was ubiquitinated and rapidly degraded in vivo. Lys-substituted versions of IAA1 localized to the nucleus as Yellow Fluorescent Protein fusions and interacted with both TIR1 and IAA7 in yeast (Saccharomyces cerevisiae) two-hybrid experiments, indicating that these proteins were functional. Ubiquitination on both HIS(6x)-HA(3x)-IAA1 and Lys-less HIS(6x)-HA(3x)-IAA1 proteins was sensitive to sodium hydroxide treatment, indicative of ubiquitin oxyester formation on serine or threonine residues. Additionally, base-resistant forms of ubiquitinated IAA1 were observed for HIS(6x)-HA(3x)-IAA1, suggesting additional lysyl-linked ubiquitin on this protein. Characterization of other Aux/IAA proteins showed that they have diverse degradation rates, adding additional complexity to auxin signaling. Altogether, these data

  9. Role of methionine in biosynthesis of prodigiosin by Serratia marcescens.

    PubMed

    Qadri, S M; Williams, R P

    1973-12-01

    Methionine alone did not allow biosynthesis of prodigiosin (2-methyl-3-amyl-6-methoxyprodigiosene) in nonproliferating cells (NPC) of Serratia marcescens strain Nima. However, when methionine was added to NPC synthesizing prodigiosin in the presence of other amino acids, the lag period for synthesis of prodigiosin was shortened, an increased amount of the pigment was formed, and the optimal concentrations of the other amino acids were reduced. Less prodigiosin was synthesized when addition of methionine was delayed beyond 4 h. The specific activity of prodigiosin synthesized by addition of (14)CH(3)-methionine was 40 to 50 times greater than that synthesized from methionine-2-(14)C or (14)COOH-methionine. NPC of mutant OF of S. marcescens synthesized norprodigiosin (2-methyl-3-amyl-6-hydroxyprodigiosene), and the specific activity of this pigment synthesized in the presence of (14)CH(3)-methionine was only 5 to 13 times greater than that synthesized from methionine-2-(14)C or (14)COOH-methionine. A particulate, cell-free extract of mutant WF of S. marcescens methylated norprodigiosin to form prodigiosin. When the extract was added to NPC of mutant OF synthesizing norprodigiosin in the presence of (14)CH(3)-methionine, the prodigiosin formed had 80% greater specific activity than the norprodigiosin synthesized in the absence of the extract. The C6 hydroxyl group of norprodigiosin was methylated in the presence of the extract and methionine. Biosynthesis of prodigiosin by NPC of strain Nima also was augmented by addition of S-adenosylmethionine. Various analogues of methionine such as norleucine, norvaline, ethionine, and alpha-methylmethionine did not affect biosynthesis of prodigiosin by NPC either in the presence or absence of methionine.

  10. Cellular and Subcellular Localization of S-Adenosyl-l-Methionine:Benzoic Acid Carboxyl Methyltransferase, the Enzyme Responsible for Biosynthesis of the Volatile Ester Methylbenzoate in Snapdragon Flowers1

    PubMed Central

    Kolosova, Natalia; Sherman, Debra; Karlson, Dale; Dudareva, Natalia

    2001-01-01

    The benzenoid ester, methylbenzoate is one of the most abundant scent compounds detected in the majority of snapdragon (Antirrhinum majus) varieties. It is produced in upper and lower lobes of petals by enzymatic methylation of benzoic acid in the reaction catalyzed by S-adenosyl-l-methionine:benzoic acid carboxyl methyltransferase (BAMT). To identify the location of methylbenzoate biosynthesis, we conducted an extensive immunolocalization study by light and electron microscopy at cellular and subcellular levels using antibodies against BAMT protein. BAMT was immunolocalized predominantly in the conical cells of the inner epidermal layer and, to a much lesser extent, in the cells of the outer epidermis of snapdragon flower petal lobes. It was also located in the inner epidermis of the corolla tube with little BAMT protein detected in the outer epidermis and in the yellow hairs within the tube on the bee's way to the nectar. These results strongly suggest that scent biosynthetic genes are expressed almost exclusively in the epidermal cells of floral organs. Immunogold labeling studies reveal that BAMT is a cytosolic enzyme, suggesting cytosolic location of methylbenzoate biosynthesis. The concentration of scent production on flower surfaces that face the pollinators during landing may increase pollination efficiency and also help to minimize the biosynthetic cost of advertising for pollinators. PMID:11457946

  11. Transcriptional and post-translational regulation of S-adenosyl-L-methionine: salicylic acid carboxyl methyltransferase (SAMT) during Stephanotis floribunda flower development.

    PubMed

    Pott, Marcella B; Effmert, Uta; Piechulla, Birgit

    2003-06-01

    Methyl salicylate (MeSA) and a number of other volatiles are primarily emitted in the evening/night by Stephanotis floribunda leading to attraction of night active pollinators. A second minor emission peak for MeSA occurs in the morning/day. To understand these emission patterns, we have studied in detail the temporal regulation of the last step of the biosynthetic pathway of MeSA, the convertion of salicylic acid (SA) to MeSA catalysed by S-adenosyl-L-methionine: salicylic acid carboxyl methyltransferase (SAMT). We observed that in young flowers a maximum in SAMT activity occurs in the night, and that in flowers which were open longer than 4 days, two SAMT activity maxima occurred per day. These maxima correlated well with dawn and dusk and the previously detected MeSA emission peaks. The SAMT mRNA levels, however, have a broad maximum during the dark phase, while the SAMT protein levels continuously increase during floral development without showing daily rhythms. Furthermore, under continuous illumination (LL) the SAMT mRNA levels and activity patterns oscillate, suggesting the involvement of a circadian clock in the regulation network. Taken together, this analysis clearly demonstrates that regulation of MeSA emission occurs both at the transcriptional and post-translational levels, indicating that control at more than one level is necessary to guarantee the precise timing of volatile emission in flowers of S. floribunda. PMID:12872485

  12. Bio-inspired nitrile hydration by peptidic ligands based on L-cysteine, L-methionine or L-penicillamine and pyridine-2,6-dicarboxylic acid.

    PubMed

    Byrne, Cillian; Houlihan, Kate M; Devi, Prarthana; Jensen, Paul; Rutledge, Peter J

    2014-01-01

    Nitrile hydratase (NHase, EC 4.2.1.84) is a metalloenzyme which catalyses the conversion of nitriles to amides. The high efficiency and broad substrate range of NHase have led to the successful application of this enzyme as a biocatalyst in the industrial syntheses of acrylamide and nicotinamide and in the bioremediation of nitrile waste. Crystal structures of both cobalt(III)- and iron(III)-dependent NHases reveal an unusual metal binding motif made up from six sequential amino acids and comprising two amide nitrogens from the peptide backbone and three cysteine-derived sulfur ligands, each at a different oxidation state (thiolate, sulfenate and sulfinate). Based on the active site geometry revealed by these crystal structures, we have designed a series of small-molecule ligands which integrate essential features of the NHase metal binding motif into a readily accessible peptide environment. We report the synthesis of ligands based on a pyridine-2,6-dicarboxylic acid scaffold and L-cysteine, L-S-methylcysteine, L-methionine or L-penicillamine. These ligands have been combined with cobalt(III) and iron(III) and tested as catalysts for biomimetic nitrile hydration. The highest levels of activity are observed with the L-penicillamine ligand which, in combination with cobalt(III), converts acetonitrile to acetamide at 1.25 turnovers and benzonitrile to benzamide at 1.20 turnovers.

  13. Suppression of human cervical cancer cell lines Hela and DoTc2 4510 by a mixture of lysine, proline, ascorbic acid, and green tea extract.

    PubMed

    Roomi, M W; Ivanov, V; Kalinovsky, T; Niedzwiecki, A; Rath, M

    2006-01-01

    Cervical cancer, the second most common cancer in women, once metastasized, leads to poor prognosis. We investigated the antitumor effect of a nutrient mixture (NM) containing lysine, proline, arginine, ascorbic acid, and green tea extract on human cervical cancer cells Hela (CCL-2) and DoTc2 4510 by measuring cell proliferation (MTT assay), modulation of matrix metalloproteinases (MMP)-2 and MMP-9) expression (gelatinase zymography), and cancer cell invasive potential (Matrigel). NM showed significant antiproliferative effect on CCL-2 and DoTc2 4510 cancer cells. The NM inhibited CCL-2 expression of MMP-2 and MMP-9 in a dose-dependent fashion, with virtual total inhibition of MMP-2 at 1000 microg/mL and MMP-9 at 500 microg/mL NM. Untreated DoTc2 4510 cells showed MMP-9 expression, which was enhanced with phorbol 12-myristate 13-acetate treatment. NM inhibited MMP-9 expression in a dose-dependent fashion, with virtual inhibition at 500 microg/mL. Invasion of human cervical cancer cells CCL-2 and DoTc2 4510 through Matrigel decreased in a dose-dependent fashion, with 100% inhibition at 500 microg/mL NM (P < 0.0001) and 1000 microg/mL NM (P < 0.0001), respectively. Our results suggest that the mixture of lysine, proline, arginine, ascorbic acid, and green tea extract has potential in the treatment of cervical cancer by inhibiting critical steps in cancer development and spread.

  14. Experimental and theoretical proton affinities of methionine, methionine sulfoxide and their N- and C-terminal derivatives

    NASA Astrophysics Data System (ADS)

    Lioe, Hadi; O'Hair, Richard A. J.; Gronert, Scott; Austin, Allen; Reid, Gavin E.

    2007-11-01

    The proton affinities of methionine, methionine sulfoxide and their derivatives (methionine methyl ester, methionine sulfoxide methyl ester, methionine methyl amide, methionine sulfoxide methyl amide, N-acetyl methionine, N-acetyl methionine sulfoxide, N-acetyl methionine methyl ester, N-acetyl methionine sulfoxide methyl ester, N-acetyl methionine methyl amide and N-acetyl methionine sulfoxide methyl amide) were experimentally determined using the kinetic method, in which proton bound dimers formed via electrospray ionization (ESI) were subjected to collision induced dissociation (CID) in a triple quadrupole mass spectrometer. In addition, theoretical calculations carried out at the MP2/6-311 + G(2d,p)//B3LYP/6-31 + G(d,p) level of theory to determine the global minima of the neutral and protonated species of all derivatives studied, were used to predict theoretical proton affinities. The density function theory calculations not only support the experimental proton affinities, but also provide structural insights into the types of hydrogen bonding that stabilize the neutral and protonated methionine or methionine sulfoxide derivatives. Comparison of the proton affinities of the various methionine and methionine sulfoxide derivatives reveals that: (i) oxidation of methionine derivatives to methionine sulfoxide derivatives results in an increase in proton affinity due to higher intrinsic proton affinity and an increase in the ring size formed through charge complexation of the sulfoxide group, which allows more efficient hydrogen bonding compared to the sulfide group; (ii) C-terminal modification by methyl esterification or methyl amidation increases the proton affinity in the order of methyl amide > methyl ester > carboxylic acid due to improved charge stabilization; (iii) N-terminal modification by N-acetylation decreases proton affinity of the derivatives due to lower intrinsic proton affinity of the N-acetyl group as well as due to stabilization of the attached

  15. Quantitation of sulfur-containing amino acids, homocysteine, methionine and cysteine in dried blood spot from newborn baby by HPLC-fluorescence detection.

    PubMed

    Wada, Mitsuhiro; Kuroki, Mana; Minami, Yuu; Ikeda, Rie; Sekitani, Yui; Takamura, Noboru; Kawakami, Shigeru; Kuroda, Naotaka; Nakashima, Kenichiro

    2014-06-01

    Sulfur-containing amino acids (SAAs), homocysteine (Hcy), methionine (Met) and cysteine (Cys) in blood are related to homocystinuria, an inborn error of metabolism. In this study, an assay method with HPLC-fluorescence detection to quantify the SAAs in a dried blood spot was established and applied to samples from newborn babies (n=200). Sample pretreatment involving reduction, derivatization with 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole, and liquid-liquid extraction with ethyl acetate gave the separation of the derivatives with retention times within 12 min. The method was enough sensitive to determine the SAAs in a dried blood spot with 0.04-0.14 µm as the limit of detection at a signal-to-noise ratio of 3. However, the absolute recoveries were very low (5.7% for Hcy, 4.6% for Cys) except for Met (105.4%) owing to inefficient recovery of Hcy and Cys from the blood matrix. Other validation parameters such as accuracy (93.5-106.2%) and intra- (≤ 9.0%) and inter-day precisions (≤ 8.7%) were acceptable. The reliability of a dried blood spot as an analytical sample was estimated. Furthermore, the proposed method was successfully applied to dried blood spots prepared from newborn babies.

  16. Geochemical behaviour of palladium in soils and Pd/PdO model substances in the presence of the organic complexing agents L-methionine and citric acid.

    PubMed

    Zereini, Fathi; Wiseman, Clare L S; Vang, My; Albers, Peter; Schneider, Wolfgang; Schindl, Roland; Leopold, Kerstin

    2016-01-01

    Risk assessments of platinum group metal (PGE) emissions, notably those of platinum (Pt), palladium (Pd) and rhodium (Rh), have been mostly based on data regarding the metallic forms used in vehicular exhaust converters, known to be virtually biologically inert and immobile. To adequately assess the potential impacts of PGE, however, data on the chemical behaviour of these metals under ambient conditions post-emission is needed. Complexing agents with a high affinity for metals in the environment are hypothesized to contribute to an increased bioaccessibility of PGE. The purpose of this study is to examine the modulating effects of the organic complexing agents, L-methionine and citric acid, on the geochemical behavior of Pd in soils and model substances (Pd black and PdO). Batch experimental tests were conducted with soils and model substances to examine the impacts of the concentration of complexing agents, pH and length of extraction period on Pd solubility and its chemical transformation. Particle surface chemistry was examined using X-ray photoelectron spectroscopy (XPS) on samples treated with solutions under various conditions, including low and high O2 levels. Pd was observed to be more soluble in the presence of organic complexing agents, compared to Pt and Rh. Pd in soils was more readily solubilized with organic complexing agents compared to the model substances. After 7 days of extraction, L-methionine (0.1 M) treated soil and Pd black samples, for instance, had mean soluble Pd fractions of 12.4 ± 5.9% and 0.554 ± 0.024%, respectively. Surface chemistry analyses (XPS) confirmed the oxidation of metallic Pd surfaces when treated with organic complexing agents. The type of organic complexing agent used for experimental purposes was observed to be the most important factor influencing solubility, followed by solution pH and time of extraction. The results demonstrate that metallic Pd can be transformed into more bioaccessible species in the presence of

  17. A comparative study of two novel nanosized radiolabeled analogues of methionine for SPECT tumor imaging.

    PubMed

    Khosroshahi, A G; Amanlou, M; Sabzevari, O; Daha, F J; Aghasadeghi, M R; Ghorbani, M; Ardestani, M S; Alavidjeh, M S; Sadat, S M; Pouriayevali, M H; Mousavi, L; Ebrahimi, S E S

    2013-01-01

    It has been reported that most tumor cells show an increased uptake of variety of amino acids specially methionine when compared with normal cells and amino acid transport is generally increased in malignant transformation. Based on the evidences, two novel nanosized analogues of methionine (Anionic Linear Globular Dendrimer G(2), a biodigredabale anionic linear globular-Methionin, and DTPA-Methionine(1) conjugates) were synthesized and labeled with (99m)Tc and used in tumor imaging/ therapy in vitro and in vivo. The results showed marked tumor SPECT molecular imaging liabilities for both compounds but with a better performance by administration of (99m)Tc-Dendrimer G(2)-Methionin. The results also showed a good anticancer activity for 99mTc-DTPA-Methionine. Based on the present study (99m)Tc-Dendrimer G(2)-Methionin or 99mTc-DTPA-(Methionine)(1) have potentials to be used in tumor molecular imaging as well as cancer therapy in future.

  18. Lysine and novel hydroxylysine lipids in soil bacteria: amino acid membrane lipid response to temperature and pH in Pseudopedobacter saltans

    PubMed Central

    Moore, Eli K.; Hopmans, Ellen C.; Rijpstra, W. Irene C.; Sánchez-Andrea, Irene; Villanueva, Laura; Wienk, Hans; Schoutsen, Frans; Stams, Alfons J. M.; Sinninghe Damsté, Jaap S.

    2015-01-01

    Microbial decomposition of organic matter is an essential process in the global carbon cycle. The soil bacteria Pseudopedobacter saltans and Flavobacterium johnsoniae are both able to degrade complex organic molecules, but it is not fully known how their membrane structures are adapted to their environmental niche. The membrane lipids of these species were extracted and analyzed using high performance liquid chromatography-electrospray ionization/ion trap/mass spectrometry (HPLC-ESI/IT/MS) and high resolution accurate mass/mass spectrometry (HRAM/MS). Abundant unknown intact polar lipids (IPLs) from P. saltans were isolated and further characterized using amino acid analysis and two dimensional nuclear magnetic resonance (NMR) spectroscopy. Ornithine IPLs (OLs) with variable (hydroxy) fatty acid composition were observed in both bacterial species. Lysine-containing IPLs (LLs) were also detected in both species and were characterized here for the first time using HPLC-MS. Novel LLs containing hydroxy fatty acids and novel hydroxylysine lipids with variable (hydroxy) fatty acid composition were identified in P. saltans. The confirmation of OL and LL formation in F. johnsoniae and P. saltans and the presence of OlsF putative homologs in P. saltans suggest the OlsF gene coding protein is possibly involved in OL and LL biosynthesis in both species, however, potential pathways of OL and LL hydroxylation in P. saltans are still undetermined. Triplicate cultures of P. saltans were grown at three temperature/pH combinations: 30°C/pH 7, 15°C/pH 7, and 15°C/pH 9. The fractional abundance of total amino acid containing IPLs containing hydroxylated fatty acids was significantly higher at higher temperature, and the fractional abundance of lysine-containing IPLs was significantly higher at lower temperature and higher pH. These results suggest that these amino acid-containing IPLs, including the novel hydroxylysine lipids, could be involved in temperature and pH stress

  19. Selective solid-phase isolation of methionine-containing peptides and subsequent matrix-assisted laser desorption/ionisation mass spectrometric detection of methionine- and of methionine-sulfoxide-containing peptides.

    PubMed

    Grunert, Tom; Pock, Katharina; Buchacher, Andrea; Allmaier, Günter

    2003-01-01

    Methionine residues and the oxidised forms in proteins are becoming more and more important in view of their biological function. In particular, methionine sulfoxide seems to have a regulatory function. This paper presents a fast strategy for simultaneous determination of methionine- and methionine-sulfoxide-containing peptides, involving application of methionine-specific solid-phase reagent chemistry combined with matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS). In the first step, methionine-containing peptides are covalently bound as sulfonium salts to glass beads, whereas methionine-sulfoxide-containing peptides and other methionine-free peptides are not bound and are washed out. The wash solution is used for MALDI-MS analysis to determine the molecular masses of these peptides and to perform, if necessary, seamless post-source decay (PSD) fragment ion analysis. Methionine-sulfoxide-containing peptides can be identified due to the characteristic metastable loss of methanesulfenic acid from the protonated molecules. In the second step, the bound peptides are cleaved from the matrix of the beads by addition of 2-mercaptoethanol at pH 8.5-8.8. The resulting peptides, mainly methionine-containing peptides, are analysed in a straightforward manner by MALDI-MS and seamless PSD. The strategy allows the fast identification of methionine- and methionine-sulfoxide-containing peptides even in complex tryptic digests, as demonstrated here for the glycoprotein antithrombin. These results show that sometimes methionine-containing tryptic peptides are not detected due to steric restrictions (e.g. glycosylation near the methionine residue) on the binding reaction, and that, on the other hand, some methionine-free peptides can be quite strongly bound non-covalently to the matrix of the beads. The latter observation indicates the necessity of seamless PSD fragment ion analysis for unambiguous identification. Furthermore, there are indications that

  20. Copper utilization in humans as affected by amino acid supplements

    SciTech Connect

    Kies, C.; Chuang, J.H.; Fox, H.M. )

    1989-02-09

    Earlier work suggests that absorption of copper as well as several other mineral nutrients may be promoted, inhibited or unaffected by the formation of mineral-amino acid complexes. The objective of the current project was to determine effects of low level supplements of selected amino acids on copper utilization. In a series of studies, healthy, human adult subjected received a basal diet with or without test supplements in separate 14-day periods which were arranged according to a randomized, cross-over design. Test amino acids and amounts given per subject per day were as follows; L-arginine, 1.2 g; L-lysine, 1.0 g; L-cystine, 1.0 g and L-methionine, 1.0 g. Subjects made complete collections of urine and stools. Fasting blood samples were drawn. Food, urine, feces and blood were analyzed for copper contents using a carbon rod attachment on a Varian atomic absorption spectrophotometer. Fecal copper losses were unaffected by used of lysine, tryptophan and methionine supplements but were reduced with use of the arginine and cystine supplements. Urine losses of copper were reduced with used of the lysine and tryptophan supplements, were increased with the methionine and cystine supplements and were unaffected when the arginine supplements were employed. Blood serum copper levels were not significantly affected by use of these supplement although some trends were noted.

  1. X-ray studies of crystalline complexes involving amino acids and peptides. XLIV. Invariant features of supramolecular association and chiral effects in the complexes of arginine and lysine with tartaric acid.

    PubMed

    Selvaraj, M; Thamotharan, S; Roy, Siddhartha; Vijayan, M

    2007-06-01

    The tartaric acid complexes with arginine and lysine exhibit two stoichiometries depending upon the ionization state of the anion. The structures reported here are DL-argininium DL-hydrogen tartrate, bis(L-argininium) L-tartrate, bis(DL-lysinium) DL-tartrate monohydrate, L-lysinium D-hydrogen tartrate and L-lysinium L-hydrogen tartrate. During crystallization, L-lysine preferentially interacts with D-tartaric acid to form a complex when DL-tartaric acid is used in the experiment. The anions and the cations aggregate into separate alternating layers in four of the five complexes. In bis(L-argininium) L-tartrate, the amino acid layers are interconnected by individual tartrate ions which do not interact among themselves. The aggregation of argininium ions in the DL- and the L-arginine complexes is remarkably similar, which is in turn similar to those observed in other dicarboxylic acid complexes of arginine. Thus, argininium ions have a tendency to assume similar patterns of aggregation, which are largely unaffected by a change in the chemistry of partner molecules such as the introduction of hydroxyl groups or a change in chirality or stoichiometry. On the contrary, the lysinium ions exhibit fundamentally different aggregation patterns in the DL-DL complexes on the one hand and L-D and L-L complexes on the other. Interestingly, the pattern in the L-D complex is similar to that in the L-L complex. The lysinium ions in the DL-DL complex exhibit an aggregation pattern similar to those observed in the DL-lysine complexes involving other dicarboxylic acids. Thus, the effect of change in the chirality of a subset of the component complexes could be profound or marginal, in an unpredictable manner. The relevant crystal structures appear to indicate that the preference of L-lysine for D-tartaric acid is perhaps caused by chiral discrimination resulting from the amplification of a small energy difference.

  2. Estimation of the optimal standardized ileal digestible lysine requirement for primiparous lactating sows fed diets supplemented with crystalline amino acids.

    PubMed

    Shi, Meng; Zang, Jianjun; Li, Zhongchao; Shi, Chuanxin; Liu, Ling; Zhu, Zhengpeng; Li, Defa

    2015-10-01

    This experiment was conducted to determine the optimal standardized ileal digestible lysine (SID Lys) level in diets fed to primiparous sows during lactation. A total of 150 (Landrace × Large White) crossbred gilts (weighing 211.1 ± 3.5 kg with a litter size of 11.1 ± 0.2) were fed lactation diets (3325 kcal metabolizable energy (ME)/kg) containing SID Lys levels of 0.76, 0.84, 0.94, 1.04 or 1.14%, through 28 days lactation. Gilts were allocated to treatments based on their body weight and backfat thickness 48 h after farrowing. Gilt body weight loss was significantly (P < 0.05) decreased by increasing dietary SID Lys levels. Fitted broken-line (P < 0.05) and quadratic plot (P < 0.05) analysis of body weight loss indicated that the optimal SID Lys for primiparous sows was 0.85 and 1.01%, respectively. Average daily feed intake (ADFI), weaning-to-estrus interval and subsequent conception rate were not affected by dietary SID Lys levels. Increasing dietary lysine had no effect on litter performances. Protein content in milk was increased by dietary SID Lys (P < 0.05). Dietary SID Lys tended to increase concentrations of serum insulin-like growth factor I (P = 0.066). These results of this experiment indicate that the optimal dietary SID Lys for lactating gilts was at least 0.85%, which approaches the recommendation of 0.84% that is estimated by the National Research Council (2012).

  3. NMR studies of protonation and hydrogen bond states of internal aldimines of pyridoxal 5'-phosphate acid-base in alanine racemase, aspartate aminotransferase, and poly-L-lysine.

    PubMed

    Chan-Huot, Monique; Dos, Alexandra; Zander, Reinhard; Sharif, Shasad; Tolstoy, Peter M; Compton, Shara; Fogle, Emily; Toney, Michael D; Shenderovich, Ilya; Denisov, Gleb S; Limbach, Hans-Heinrich

    2013-12-01

    Using (15)N solid-state NMR, we have studied protonation and H-bonded states of the cofactor pyridoxal 5'-phosphate (PLP) linked as an internal aldimine in alanine racemase (AlaR), aspartate aminotransferase (AspAT), and poly-L-lysine. Protonation of the pyridine nitrogen of PLP and the coupled proton transfer from the phenolic oxygen (enolimine form) to the aldimine nitrogen (ketoenamine form) is often considered to be a prerequisite to the initial step (transimination) of the enzyme-catalyzed reaction. Indeed, using (15)N NMR and H-bond correlations in AspAT, we observe a strong aspartate-pyridine nitrogen H-bond with H located on nitrogen. After hydration, this hydrogen bond is maintained. By contrast, in the case of solid lyophilized AlaR, we find that the pyridine nitrogen is neither protonated nor hydrogen bonded to the proximal arginine side chain. However, hydration establishes a weak hydrogen bond to pyridine. To clarify how AlaR is activated, we performed (13)C and (15)N solid-state NMR experiments on isotopically labeled PLP aldimines formed by lyophilization with poly-L-lysine. In the dry solid, only the enolimine tautomer is observed. However, a fast reversible proton transfer involving the ketoenamine tautomer is observed after treatment with either gaseous water or gaseous dry HCl. Hydrolysis requires the action of both water and HCl. The formation of an external aldimine with aspartic acid at pH 9 also produces the ketoenamine form stabilized by interaction with a second aspartic acid, probably via a H-bond to the phenolic oxygen. We postulate that O-protonation is an effectual mechanism for the activation of PLP, as is N-protonation, and that enzymes that are incapable of N-protonation employ this mechanism. PMID:24147985

  4. Lysine catabolism in Rhizoctonia leguminicola and related fungi.

    PubMed Central

    Guengerich, F P; Broquist, H P

    1976-01-01

    The catabolism of lysine was studied in several yeasts and fungi. Results with cell-free extracts of Rhizoctonia leguminicola support a proposed pathway involving (D- and L-) EPSILON-N-acetyllysine, alpha-keto-epsilon-acetamidohexanoic acid, delta-acetamidovaleric acid, and delta-aminovaleric acid in the conversion of L-lysine to shortchain organic acids. Label from radioactive L-lysine was found to accumulate in D- and L-epsilon-N-acetyllysine, delta-acetamidovaleric acid, delta-aminovaleric acid, and glutaric acid in cultures of R. leguminicola, Neurospora crassa, Saccharomyces cerevisiae, and Hansenula saturnus, suggesting that the proposed omega-acetyl pathway of lysine catabolism is generalized among yeasts and fungi. In N. crassa, as is the case in R. leguminicola, the major precursor of L-pipecolic acid was the L-isomer of lysine; 15N experiments were consistent with delta1-piperideine-2-carboxylic acid as an intermediate in the transformation. PMID:131119

  5. Intestinal absorption of amino acids in the Pacific bluefin tuna (Thunnus orientalis): in vitro lysine-arginine interaction using the everted intestine system.

    PubMed

    Martínez-Montaño, Emmanuel; Peña, Emyr; Viana, María Teresa

    2013-04-01

    The interaction between lysine (Lys) and arginine (Arg) in the proximal intestinal region of Pacific bluefin tuna (Thunnus orientalis) was evaluated using the everted intestine method. This in vitro intestinal system has been shown to be an effective tool for studying the nutrient absorption without the need to handle the tuna fish in marine cages as needed for digestibility and amino acid (AA) absorption. We used a factorial design with two sets of variables: low and high Lys concentration (10 and 75 mM) and four different Arg concentrations (3, 10, 20, and 30 mM). Both amino acids were dissolved in marine Ringer solution with a basal amino acidic composition consisting of a tryptone solution (9 mg mL(-1)). No interaction was observed between the absorption of Lys and Arg during the first 10 min of the experiment when low concentration of Lys and Arg was used in the hydrolyzate solution. However, there seemed to be a positive effect on Lys absorption when both amino acids were at high concentrations (30 and 75 mM, respectively). This type of studies will led us to test different formulations and/or additives to better understand the efficiency of AA supplementation as an alternative to in situ studies that are difficult to follow to design with the Pacific Bluefin Tuna.

  6. A Methionine Residue Promotes Hyperoxidation of the Catalytic Cysteine of Mouse Methionine Sulfoxide Reductase A.

    PubMed

    Kim, Geumsoo; Levine, Rodney L

    2016-06-28

    Methionine sulfoxide reductase A (msrA) reduces methionine sulfoxide in proteins back to methionine. Its catalytic cysteine (Cys72-SH) has a low pKa that facilitates oxidation by methionine sulfoxide to cysteine sulfenic acid. If the catalytic cycle proceeds efficiently, the sulfenic acid is reduced back to cysteine at the expense of thioredoxin. However, the sulfenic acid is vulnerable to "irreversible" oxidation to cysteine sulfinic acid that inactivates msrA (hyperoxidation). We observed that human msrA is resistant to hyperoxidation while mouse msrA is readily hyperoxidized by micromolar concentrations of hydrogen peroxide. We investigated the basis of this difference in susceptibility to hyperoxidation and established that it is controlled by the presence or absence of a Met residue in the carboxyl-terminal domain of the enzyme, Met229. This residue is Val in human msrA, and when it was mutated to Met, human msrA became sensitive to hyperoxidation. Conversely, mouse msrA was rendered insensitive to hyperoxidation when Met229 was mutated to Val or one of five other residues. Positioning of the methionine at residue 229 is not critical, as hyperoxidation occurred as long as the methionine was located within the group of 14 carboxyl-terminal residues. The carboxyl domain of msrA is known to be flexible and to have access to the active site, and Met residues are known to form stable, noncovalent bonds with aromatic residues through interaction of the sulfur atom with the aromatic ring. We propose that Met229 forms such a bond with Trp74 at the active site, preventing formation of a protective sulfenylamide with Cys72 sulfenic acid. As a consequence, the sulfenic acid is available for facile, irreversible oxidation to cysteine sulfinic acid. PMID:27259041

  7. Engineering of methionine chain elongation part of glucoraphanin pathway in E. coli.

    PubMed

    Mirza, Nadia; Crocoll, Christoph; Erik Olsen, Carl; Ann Halkier, Barbara

    2016-05-01

    The methionine-derived glucosinolate glucoraphanin is associated with the health-promoting properties of broccoli. This has developed a strong interest in producing this compound in high amounts from a microbial source. Glucoraphanin synthesis starts with a five-gene chain elongation pathway that converts methionine to dihomo-methionine, which is subsequently converted to glucoraphanin by the seven-gene glucosinolate core structure pathway. As dihomo-methionine is the precursor amino acid for glucoraphanin production, a first challenge is to establish an expression system for production of dihomo-methionine. In planta, the methionine chain elongation enzymes are physically separated within the cell with the first enzyme in the cytosol while the rest are located in the chloroplast. A de-compartmentalization approach was applied to produce dihomo-methionine by expression of the respective plant genes in Escherichia coli cytosol. Introduction of two plasmids encoding the methionine chain elongation pathway into E. coli resulted in production of 25mgL(-1) of dihomo-methionine. In addition to chain-elongated methionine products, side-products from chain elongation of leucine were produced. Methionine supplementation enhanced dihomo-methionine production to 57mgL(-1), while keeping a steady level of the chain-elongated leucine products. Engineering of the de-compartmentalized pathway of dihomo-methionine in E. coli cytosol provides an important first step for microbial production of the health-promoting glucoraphanin.

  8. Bioavailability of lysine in Maillard browned protein as determined by plasma lysine response in rainbow trout (Salmo gairdneri).

    PubMed

    Plakas, S M; Lee, T C; Wolke, R E

    1988-01-01

    The bioavailability of lysine in Maillard browned protein was investigated by plasma lysine response in rainbow trout (Salmo gairdneri). The concentrations of free lysine in the plasma were measured after feeding control and browned protein diets supplemented with graded levels of lysine. Bioavailability of lysine was estimated based on the amounts of supplemental lysine in the diets that resulted in rapid increases in plasma lysine. An approximately 80% loss in bioavailable lysine content was determined by this method in a fish protein isolate subjected to the Maillard browning reaction under mild conditions (40 d incubation at 37 degrees C). The nutritional damage to lysine determined by plasma lysine response was similar to that estimated in vitro by enzymatic hydrolysis and fluorodinitrobenzene reagent, but was underestimated by acid hydrolysis and trinitrobenzene sulfonic acid reagent. Rainbow trout are similar to other animals in their inability to utilize the deoxyketosyl (Amadori) compound of lysine formed in early Maillard reaction, and in their plasma response to dietary levels of essential amino acids. PMID:3121813

  9. Bioavailability of free lysine and protein-bound lysine from casein and fishmeal in juvenile turbot (Psetta maxima).

    PubMed

    Kroeckel, Saskia; Dietz, Carsten; Schulz, Carsten; Susenbeth, Andreas

    2015-03-14

    In the present study, a linear regression analysis between lysine intake and lysine retention was conducted to investigate the efficiency of lysine utilisation (k(Lys)) at marginal lysine intake of either protein-bound or free lysine sources in juvenile turbot (Psetta maxima). For this purpose, nine isonitrogenous and isoenergetic diets were formulated to contain 2·25-4·12 g lysine/100 g crude protein (CP) to ensure that lysine was the first-limiting amino acid in all diets. The basal diet contained 2·25 g lysine/100 g CP. Graded levels of casein (Cas), fishmeal (FM) and L-lysine HCl (Lys) were added to the experimental diets to achieve stepwise lysine increments. A total of 240 fish (initial weight 50·1 g) were hand-fed all the experimental diets once daily until apparent satiation over a period of 56 d. Feed intake was significantly affected by dietary lysine concentration rather than by dietary lysine source. Specific growth rate increased significantly at higher lysine concentrations (P< 0·001). CP, crude lipid and crude ash contents in the whole body were affected by the dietary treatments. The linear regression slope between lysine retention and lysine intake (k(Lys)) was similar between all the dietary lysine sources. The k(Lys) values for the diets supplemented with Cas, Lys or FM were 0·833, 0·857 and 0·684, respectively. The bioavailability of lysine from the respective lysine sources was determined by a slope-ratio approach. The bioavailability of lysine (relative to the reference lysine source Cas) from FM and Lys was 82·1 and 103 %, respectively. Nutrient requirement for maintenance was in the range of 16·7-23·4 mg/kg(0·8) per d, and did not differ between the treatments. There were no significant differences in lysine utilisation efficiency or bioavailability of protein-bound or crystalline lysine from the respective sources observed when lysine was confirmed to be the first-limiting nutrient.

  10. Limiting amino acid for protein synthesis with mammary cells in tissue culture.

    PubMed

    Park, C S; Chandler, P T; Norman, A W

    1976-05-01

    To identify the limiting amino acid in the minimal essential medium as published by Eagle (Science 130:432, 1959) for milk protein synthesis in rat mammary cells in tissue culture, two different experimental approaches were used. The first study involved the reduction of amino acids singly from the total amino acid complement of the medium for milk protein synthesis. The second study was to investigate the effect on milk protein synthesis of single amino acid addition to the basic complement of amino acids. Order of limiting amino acids was lysine (first) and possible methionine, valine, or arginine (second).

  11. Genetic identification of ACC-RESISTANT2 reveals involvement of LYSINE HISTIDINE TRANSPORTER1 in the uptake of 1-aminocyclopropane-1-carboxylic acid in Arabidopsis thaliana.

    PubMed

    Shin, Kihye; Lee, Sumin; Song, Won-Yong; Lee, Rin-A; Lee, Inhye; Ha, Kyungsun; Koo, Ja-Choon; Park, Soon-Ki; Nam, Hong-Gil; Lee, Youngsook; Soh, Moon-Soo

    2015-03-01

    1-Aminocyclopropane-1-carboxylic acid (ACC) is a biosynthetic precursor of ethylene, a gaseous plant hormone which controls a myriad of aspects of development and stress adaptation in higher plants. Here, we identified a mutant in Arabidopsis thaliana, designated as ACC-resistant2 (are2), displaying a dose-dependent resistance to exogenously applied ACC. Physiological analyses revealed that mutation of are2 impaired various aspects of exogenous ACC-induced ethylene responses, while not affecting sensitivity to other plant hormones during seedling development. Interestingly, the are2 mutant was normally sensitive to gaseous ethylene, compared with the wild type. Double mutant analysis showed that the ethylene-overproducing mutations, eto1 or eto3, and the constitutive ethylene signaling mutation, ctr1 were epistatic to the are2 mutation. These results suggest that the are2 mutant is not defective in ethylene biosynthesis or ethylene signaling per se. Map-based cloning of ARE2 demonstrated that LYSINE HISTIDINE TRANSPORTER1 (LHT1), encoding an amino acid transporter, is the gene responsible. An uptake experiment with radiolabeled ACC indicated that mutations of LHT1 reduced, albeit not completely, uptake of ACC. Further, we performed an amino acid competition assay and found that two amino acids, alanine and glycine, known as substrates of LHT1, could suppress the ACC-induced triple response in a LHT1-dependent way. Taken together, these results provide the first molecular genetic evidence supporting that a class of amino acid transporters including LHT1 takes part in transport of ACC, thereby influencing exogenous ACC-induced ethylene responses in A. thaliana. PMID:25520403

  12. Oxidation of methionine in PrP is dependent upon the oxidant and the amino acid two positions removed(Abstract)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background/Introduction. Methionine oxidation has been shown both to be associated with prion formation and implicated in the inhibition of amyloid formation in model systems. This work is based on model systems where hydrogen peroxide was used as an oxidant. Materials and Methods. We developed a se...

  13. A phase transition from monoclinic C2 with Z' = 1 to triclinic P1 with Z' = 4 for the quasiracemate L-2-aminobutyric acid-D-methionine (1/1).

    PubMed

    Görbitz, Carl Henrik; Wragg, David S; Bakke, Ingrid Marie Bergh; Fleischer, Christian; Grønnevik, Gaute; Mykland, Maria; Park, Yoomin; Trovik, Kristian Wiedicke; Serigstad, Halvard; Sundsli, Bård Edgar Vestheim

    2016-07-01

    Racemates of hydrophobic amino acids with linear side chains are known to undergo a unique series of solid-state phase transitions that involve sliding of molecular bilayers upon heating or cooling. Recently, this behaviour was shown to extend also to quasiracemates of two different amino acids with opposite handedness [Görbitz & Karen (2015). J. Phys. Chem. B, 119, 4975-4984]. Previous investigations are here extended to an L-2-aminobutyric acid-D-methionine (1/1) co-crystal, C4H9NO2·C5H11NO2S. The significant difference in size between the -CH2CH3 and -CH2CH2SCH3 side chains leads to extensive disorder at room temperature, which is essentially resolved after a phase transition at 229 K to an unprecedented triclinic form where all four D-methionine molecules in the asymmetric unit have different side-chain conformations and all three side-chain rotamers are used for the four partner L-2-aminobutyric acid molecules. PMID:27377274

  14. Differential trace labeling of calmodulin: investigation of binding sites and conformational states by individual lysine reactivities. Effects of beta-endorphin, trifluoperazine, and ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid

    SciTech Connect

    Giedroc, D.P.; Sinha, S.K.; Brew, K.; Puett, D.

    1985-11-05

    The CaS -dependent association of beta-endorphin and trifluoperazine with porcine testis calmodulin, as well as the effects of removing CaS by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) treatment, were investigated by the procedure of differential kinetic labeling. This technique permitted determination of the relative rates of acylation of each of the epsilon-amino groups of the seven lysyl residues on calmodulin by (TH)acetic anhydride under the different conditions. In all cases, less than 0.52 mol of lysyl residue/mol of calmodulin was modified, thus ensuring that the labeling pattern reflects the microenvironments of these groups in the native protein. Lysines 75 and 94 were found to be the most reactive amino groups in CaS -saturated calmodulin. In the presence of CaS and under conditions where beta-endorphin and calmodulin were present at a molar ratio of 2.5:1, the amino groups of lysines 75 and 148 were significantly reduced in reactivity compared to calmodulin alone. At equimolar concentrations of peptides and proteins, essentially the same result was obtained except that the magnitudes of the perturbation of these two lysines were less pronounced. With trifluoperazine, at a molar ratio to calmodulin of 2.5:1, significant perturbations of lysines 75 and 148, as well as Lys 77, were also found. These results further substantiate previous observations of a commonality between phenothiazine and peptide binding sites on calmodulin. Lastly, an intriguing difference in CaS -mediated reactivities between lysines 75 and 77 of calmodulin is demonstrated. In the CaS -saturated form of the protein, both lysines are part of the long connecting helix between the two homologous halves of the protein.

  15. Economical production of poly(ε-l-lysine) and poly(l-diaminopropionic acid) using cane molasses and hydrolysate of streptomyces cells by Streptomyces albulus PD-1.

    PubMed

    Xia, Jun; Xu, Zhaoxian; Xu, Hong; Liang, Jinfeng; Li, Sha; Feng, Xiaohai

    2014-07-01

    Poly(ε-L-lysine) (ε-PL) and poly(L-diaminopropionic acid) (PDAP) co-production by Streptomyces albulus PD-1 from cane molasses and hydrolysate of strepyomyces cells (HSC) was investigated for the first time in this study. The optimal initial total sugar concentration of the cane molasses pretreated with sulfuric acid was determined to be 20 g L(-1), and HSC could substitute for yeast extract for ε-PL and PDAP co-production. When fed-batch fermentation was performed in 1t fermentor with pretreated cane molasses and HSC, 20.6 ± 0.5 g L(-1) of ε-PL and 5.2 ± 0.6 g L(-1) of PDAP were obtained. The amount of strepyomyces cells obtained in one fed-batch fermentation is sufficient to prepare the HSC to satisfy the demand of subsequent fermentations, thus the self-cycling of organic nitrogen source becomes available. These results suggest that the low-cost cane molasses and HSC can be used for the economical production of ε-PL and PDAP by S. albulus PD-1.

  16. Bacterial Lysine Decarboxylase Influences Human Dental Biofilm Lysine Content, Biofilm Accumulation and Sub-Clinical Gingival Inflammation

    PubMed Central

    Lohinai, Z.; Keremi, B.; Szoko, E.; Tabi, T.; Szabo, C.; Tulassay, Z.; Levine, M.

    2012-01-01

    Background Dental biofilms contain a protein that inhibits mammalian cell growth, possibly lysine decarboxylase from Eikenella corrodens. This enzyme decarboxylates lysine, an essential amino acid for dentally attached cell turnover in gingival sulci. Lysine depletion may stop this turnover, impairing the barrier to bacterial compounds. The aims of this study were to determine biofilm lysine and cadaverine contents before oral hygiene restriction (OHR), and their association with plaque index (PI) and gingival crevicular fluid (GCF) after OHR for a week. Methods Laser-induced fluorescence after capillary electrophoresis was used to determine lysine and cadaverine contents in dental biofilm, tongue biofilm and saliva before OHR and in dental biofilm after OHR. Results Before OHR, lysine and cadaverine contents of dental biofilm were similar and 10-fold greater than in saliva or tongue biofilm. After a week of OHR, the biofilm content of cadaverine increased and that of lysine decreased, consistent with greater biofilm lysine decarboxylase activity. Regression indicated that PI and GCF exudation were positively related to biofilm lysine post-OHR, unless biofilm lysine exceeded the minimal blood plasma content in which case PI was further increased but GCF exudation was reduced. Conclusions After OHR, lysine decarboxylase activity seems to determine biofilm lysine content and biofilm accumulation. When biofilm lysine exceeds minimal blood plasma content after OHR, less GCF appeared despite more biofilm. Lysine appears important for biofilm accumulation and the epithelial barrier to bacterial proinflammatory agents. Clinical Relevance Inhibiting lysine decarboxylase may retard the increased GCF exudation required for microbial development and gingivitis. PMID:22141361

  17. Effects of D-methionine or L-methionine on root hair of Brassica rapa.

    PubMed

    Hasegawa, Nobuharu; Yamaji, Yohei; Minoda, Masashi; Kubo, Motoki

    2003-01-01

    We examined the effects of D- or L-amino acids on the stimulation of Brassica rapa roots. When 6.7 microM of D-methionine (D-Met) or L-methionine (L-Met) was applied, root hair numbers increased. L-Met (above concentration of 67.0 microM) caused the tip of roots to spiral. When CoCl2 (ethylene synthesis inhibitor) was added into the medium, L-Met lost its activity but COCl2 did not inhibit the bioactivity of D-Met.

  18. The Arabidopsis LYSIN MOTIF-CONTAINING RECEPTOR-LIKE KINASE3 Regulates the Cross Talk between Immunity and Abscisic Acid Responses1[W][OPEN

    PubMed Central

    Paparella, Chiara; Savatin, Daniel Valentin; Marti, Lucia; De Lorenzo, Giulia; Ferrari, Simone

    2014-01-01

    Transmembrane receptor-like kinases characterized by the presence of one or more lysin motif (LysM) domains in the extracytoplasmic portion (LysM-containing receptor-like kinases [LYKs]) mediate recognition of symbiotic and pathogenic microorganisms in plants. The Arabidopsis (Arabidopsis thaliana) genome encodes five putative LYKs; among them, AtLYK1/CHITIN ELICITOR RECEPTOR KINASE1 is required for response to chitin and peptidoglycan, and AtLYK4 contributes to chitin perception. More recently, AtLYK3 has been shown to be required for full repression, mediated by Nod factors, of Arabidopsis innate immune responses. In this work, we show that AtLYK3 also negatively regulates basal expression of defense genes and resistance to Botrytis cinerea and Pectobacterium carotovorum infection. Enhanced resistance of atlyk3 mutants requires PHYTOALEXIN-DEFICIENT3, which is crucial for camalexin biosynthesis. The expression of AtLYK3 is strongly repressed by elicitors and fungal infection and is induced by the hormone abscisic acid (ABA), which has a negative impact on resistance against B. cinerea and P. carotovorum. Plants lacking a functional AtLYK3 also show reduced physiological responses to ABA and are partially resistant to ABA-induced inhibition of PHYTOALEXIN-DEFICIENT3 expression. These results indicate that AtLYK3 is important for the cross talk between signaling pathways activated by ABA and pathogens. PMID:24639336

  19. Biocompatibility Assessment of Polyethylene Glycol-Poly L-Lysine-Poly Lactic-Co-Glycolic Acid Nanoparticles In Vitro and In Vivo.

    PubMed

    Guo, Liting; Chen, Baoan; Liu, Ran; Xia, Guohua; Wang, Yonglu; Li, Xueming; Wei, Chen; Wang, Xuemei; Jiang, Hulin

    2015-05-01

    The present study was designed to evaluate the biocompatibility of nanoparticles polyethylene glycol (PEG)-poly L-lysine (PLL)-poly lactic-co-glycolic acid copolymer (PLGA) (PEG-PLL-PLGA) before clinical application. We applied some tests to assess the safety of PEG-PLL-PLGA nanoparticles (NPs). There was low cytotoxicity of PEG-PLL-PLGA NPs in vitro as detected by MTT assay. Cell apoptosis and intracellular accumulation of PEG-PLL-PLGA were determined by FCM assay. The apoptotic rate induced by nanoparticles and the fluorescence intensity of intracellular daunorubicin (DNR) demonstrated that DNR-PEG-PLL-PLGA could be taken up by the mouse fibroblast cells (L929 cells). Hemolysis test and micronucleus (MN) assay demonstrated that the nanoparticles have no obviously blood toxicity and genotoxicity. DNR-PEG-PLL-PLGA NPs were injected into mice through tail vein to calculate the median lethal dose (LD50), the results showed that they had a wide safe scale. Blood was taken by removing the eyeball of mice to study the influence of DNR-PEG-PLL-PLGA in hepatic and renal functions. The results revealed that there was no significant difference as compared with the control group. Interestingly, the pathologic changes of heart, liver, spleen, lung and kidney were observed in nanoparticles treated mice. Thus, this study demonstrates that PEG-PLL-PLGA NPs appear to be highly biocompatible and safe nanoparticles that can be suitable for further application in the treatment of tumor.

  20. Influence of assembling pH on the stability of poly(L-glutamic acid) and poly(L-lysine) multilayers against urea treatment.

    PubMed

    Zhou, Jie; Wang, Bo; Tong, Weijun; Maltseva, Elena; Zhang, Gang; Krastev, Rumen; Gao, Changyou; Möhwald, Helmuth; Shen, Jiacong

    2008-04-01

    Polyelectrolyte multilayers of poly(L-glutamic acid) (PGA) and poly(L-lysine) (PLL) were built up using the layer-by-layer (LbL) technique in low pH (3.6, PM3.6) and in neutral pH (7.4, PM7.4) solutions. The multilayers were then treated with a concentrated urea (one kind of denaturant for proteins and polypeptides) solution (8M) and rinsed with corresponding buffer. The buildup and treatment processes were investigated by ultraviolet visible spectroscopy and ellipsometry. The surface morphology was observed by scanning force microscopy (SFM). The inner structures were determined by X-ray reflectometry and circular dichroism spectroscopy (CD). An exponential growth of the optical mass and the layer thickness was observed for both PM3.6 and PM7.4. After urea treatment, a significant mass loss for PM3.6 was found, while no mass change was recorded for PM7.4. The dominant driving force for PM7.4 is electrostatic interaction, resulting in multilayers with an abundant beta-sheet structure, which has higher stability against urea treatment. By contrast, the dominant driving force for PM3.6 is hydrogen bonding and hydrophobic interaction, which are sensitive to the urea treatment. The mechanism is substantiated by molecular mechanics calculation. This has offered a convenient pathway to mediate the multilayer properties, which is of great importance for potential applications.

  1. The Arabidopsis LYSIN MOTIF-CONTAINING RECEPTOR-LIKE KINASE3 regulates the cross talk between immunity and abscisic acid responses.

    PubMed

    Paparella, Chiara; Savatin, Daniel Valentin; Marti, Lucia; De Lorenzo, Giulia; Ferrari, Simone

    2014-05-01

    Transmembrane receptor-like kinases characterized by the presence of one or more lysin motif (LysM) domains in the extracytoplasmic portion (LysM-containing receptor-like kinases [LYKs]) mediate recognition of symbiotic and pathogenic microorganisms in plants. The Arabidopsis (Arabidopsis thaliana) genome encodes five putative LYKs; among them, AtLYK1/CHITIN ELICITOR RECEPTOR KINASE1 is required for response to chitin and peptidoglycan, and AtLYK4 contributes to chitin perception. More recently, AtLYK3 has been shown to be required for full repression, mediated by Nod factors, of Arabidopsis innate immune responses. In this work, we show that AtLYK3 also negatively regulates basal expression of defense genes and resistance to Botrytis cinerea and Pectobacterium carotovorum infection. Enhanced resistance of atlyk3 mutants requires PHYTOALEXIN-DEFICIENT3, which is crucial for camalexin biosynthesis. The expression of AtLYK3 is strongly repressed by elicitors and fungal infection and is induced by the hormone abscisic acid (ABA), which has a negative impact on resistance against B. cinerea and P. carotovorum. Plants lacking a functional AtLYK3 also show reduced physiological responses to ABA and are partially resistant to ABA-induced inhibition of PHYTOALEXIN-DEFICIENT3 expression. These results indicate that AtLYK3 is important for the cross talk between signaling pathways activated by ABA and pathogens.

  2. Degradation of Amino Acids and Structure in Model Proteins and Bacteriophage MS2 by Chlorine, Bromine, and Ozone.

    PubMed

    Choe, Jong Kwon; Richards, David H; Wilson, Corey J; Mitch, William A

    2015-11-17

    Proteins are important targets of chemical disinfectants. To improve the understanding of disinfectant-protein reactions, this study characterized the disinfectant:protein molar ratios at which 50% degradation of oxidizable amino acids (i.e., Met, Tyr, Trp, His, Lys) and structure were observed during HOCl, HOBr, and O3 treatment of three well-characterized model proteins and bacteriophage MS2. A critical question is the extent to which the targeting of amino acids is driven by their disinfectant rate constants rather than their geometrical arrangement. Across the model proteins and bacteriophage MS2 (coat protein), differing widely in structure, methionine was preferentially targeted, forming predominantly methionine sulfoxide. This targeting concurs with its high disinfectant rate constants and supports its hypothesized role as a sacrificial antioxidant. Despite higher HOCl and HOBr rate constants with histidine and lysine than for tyrosine, tyrosine generally was degraded in preference to histidine, and to a lesser extent, lysine. These results concur with the prevalence of geometrical motifs featuring histidines or lysines near tyrosines, facilitating histidine and lysine regeneration upon Cl[+1] transfer from their chloramines to tyrosines. Lysine nitrile formation occurred at or above oxidant doses where 3,5-dihalotyrosine products began to degrade. For O3, which lacks a similar oxidant transfer pathway, histidine, tyrosine, and lysine degradation followed their relative O3 rate constants. Except for its low reactivity with lysine, the O3 doses required to degrade amino acids were as low as or lower than for HOCl or HOBr, indicating its oxidative efficiency. Loss of structure did not correlate with loss of particular amino acids, suggesting the need to characterize the oxidation of specific geometric motifs to understand structural degradation.

  3. Acid-base interactions and secondary structures of poly-L-lysine probed by 15N and 13C solid state NMR and Ab initio model calculations.

    PubMed

    Dos, Alexandra; Schimming, Volkmar; Tosoni, Sergio; Limbach, Hans-Heinrich

    2008-12-11

    The interactions of the 15N-labeled amino groups of dry solid poly-L-lysine (PLL) with various halogen and oxygen acids HX and the relation to the secondary structure have been studied using solid-state 15N and 13C CPMAS NMR spectroscopy (CP = cross polarization and MAS = magic angle spinning). For comparison, 15N NMR spectra of an aqueous solution of PLL were measured as a function of pH. In order to understand the effects of protonation and hydration on the 15N chemical shifts of the amino groups, DFT and chemical shielding calculations were performed on isolated methylamine-acid complexes and on periodic halide clusters of the type (CH3NH3(+)X(-))n. The combined experimental and computational results reveal low-field shifts of the amino nitrogens upon interaction with the oxygen acids HX = HF, H2SO4, CH3COOH, (CH3)2POOH, H3PO4, HNO3, and internal carbamic acid formed by reaction of the amino groups with gaseous CO2. Evidence is obtained that only hydrogen-bonded species of the type (Lys-NH2***H-X)n are formed in the absence of water. 15N chemical shifts are maximum when H is located in the hydrogen bond center and then decrease again upon full protonation, as found for aqueous solution at low pH. By contrast, halogen acids interact in a different way. They form internal salts of the type (Lys-NH3(+)X(-))n via the interaction of many acid-base pairs. This salt formation is possible only in the beta-sheet conformation. By contrast, the formation of hydrogen-bonded complexes can occur both in beta-sheet domains as well as in alpha-helical domains. The 15N chemical shifts of the protonated ammonium groups increase when the size of the interacting halogen anions is increased from chloride to iodide and when the number of the interacting anions is increased. Thus, the observed high-field 15N shift of ammonium groups upon hydration is the consequence of replacing interacting halogen atoms by oxygen atoms.

  4. Methionine restriction and life-span control.

    PubMed

    Lee, Byung Cheon; Kaya, Alaattin; Gladyshev, Vadim N

    2016-01-01

    Dietary restriction (DR) without malnutrition is associated with longevity in various organisms. However, it has also been shown that reduced calorie intake is often ineffective in extending life span. Selecting optimal dietary regimens for DR studies is complicated, as the same regimen may lead to different outcomes depending on genotype and environmental factors. Recent studies suggested that interventions such as moderate protein restriction with or without adequate nutrition (e.g., particular amino acids or carbohydrates) may have additional beneficial effects mediated by certain metabolic and hormonal factors implicated in the biology of aging, regardless of total calorie intake. In particular, it was shown that restriction of a single amino acid, methionine, can mimic the effects of DR and extend life span in various model organisms. We discuss the beneficial effects of a methionine-restricted diet, the molecular pathways involved, and the use of this regimen in longevity interventions.

  5. Protein-borne methionine residues as structural antioxidants in mitochondria.

    PubMed

    Schindeldecker, Mario; Moosmann, Bernd

    2015-07-01

    Methionine is an oxidant-labile amino acid whose major oxidation products, methionine sulfoxides, can be readily repaired by various NADPH-dependent methionine sulfoxide reductases. Formally, the methionine oxidation-reduction circuit could act as a cellular antioxidant system, by providing a safe sink for oxidants that might cause much more damage if reacting otherwise. This concept is supported by focal experimental evidence; however, the global importance, scope and biochemical role of protein-borne methionine as an inbuilt macromolecular antioxidant have remained incompletely defined. In analyzing proteomic methionine usage on different levels of comparison, we find that protein methionine (i) is primarily an antioxidant of mitochondria, especially of the inner mitochondrial membrane, (ii) responds strongly to respiratory demands on an evolutionary timescale, (iii) acts locally, by selectively protecting its carrier protein, and (iv) might be utilized as a molecular predictor of aerobic metabolic rate in animals, to complement traditional markers like the presence of a respiratory pigment. Our data support the idea that proteins in need of a long lifespan or acting in dangerous environments may acquire massive structural alterations aimed at increasing their resistance to oxidation. Counterintuitively though, they sometimes do so by accumulating particularly labile rather than particularly stable building blocks, illustrating that the technical concept of cathodic protection is also employed by the animate nature.

  6. Bioavailability of different dietary supplemental methionine sources in animals.

    PubMed

    Zhang, Shuai; Wong, Eric A; Gilbert, Elizabeth R

    2015-01-01

    Dietary methionine is indispensable for animal maintenance, growth and development. L-methionine (L-Met), and its synthetic forms DL-methionine (DL-Met) and 2-hydroxy-4 (methylthio) butanoic acid (HMTBA) are common supplemental methionine sources in animal diets. There are different characteristics for cellular absorption, transport, metabolism and bio-efficiency between these three dietary methionine sources. Moreover, there are differences in their utilization among various species such as chickens, pigs and ruminants. As a methionine precursor, HMTBA is efficacious in the promotion of growth in animals. It is absorbed mainly by monocarboxylate transporter 1 (MCT1), coupled with the activity of the Na(+)/H(+) exchanger (NHE3), while DL-Met uptake occurs via multiple carrier-mediated systems. Liver, kidney and small intestine can metabolize D-Met and HMTBA to L-Met through oxidation and transamination. In ruminants, the non-hepatic tissues act as major sites of HMTBA conversion, which are different from that in chickens and pigs. HMTBA also has additional benefits in anti-oxidation. Understanding the characteristics of uptake and metabolism of different methionine sources will greatly benefit the industry and bioscience research.

  7. Excess dietary methionine does not affect fracture healing in mice

    PubMed Central

    Holstein, Joerg H.; Schmalenbach, Julia; Herrmann, Markus; Ölkü, Ilona; Garcia, Patric; Histing, Tina; Herrmann, Wolfgang; Menger, Michael D.; Pohlemann, Tim; Claes, Lutz

    2012-01-01

    Summary Background An elevated serum concentration of homocysteine (hyperhomocysteinemia) has been shown to disturb fracture healing. As the essential amino acid, methionine, is a precursor of homocysteine, we aimed to investigate whether excess methionine intake affects bone repair. Material/Methods We analyzed bone repair in 2 groups of mice. One group was fed a methionine-rich diet (n=13), and the second group received an equicaloric control diet without methionine supplementation (n=12). Using a closed femoral fracture model, bone repair was analyzed by histomorphometry and biomechanical testing at 4 weeks after fracture. Blood was sampled to measure serum concentrations of homocysteine, the bone formation marker osteocalcin, and the bone resorption marker collagen I C-terminal crosslaps Results Serum concentrations of homocysteine were significantly higher in the methionine group than in the control group, while serum markers of bone turnover did not differ significantly between the 2 groups. Histomorphometry revealed no significant differences in size and tissue composition of the callus between animals fed the methionine-enriched diet and those receiving the control diet. Accordingly, animals of the 2 groups showed a comparable bending stiffness of the healing bones. Conclusions We conclude that excess methionine intake causes hyperhomocysteinemia, but does not affect fracture healing in mice. PMID:23197225

  8. Progression of lipid peroxidation measured as thiobarbituric acid reactive substances, damage to DNA and histopathological changes in the liver of rats subjected to a methionine-choline-deficient diet.

    PubMed

    Jordao, Alceu Afonso; Zanutto, Marcia Elena; Domenici, Fernanda Aparecida; Portari, Guilherme Vannucchi; Cecchi, Andréa Oliveira; Zucoloto, Sergio; Vannucchi, Helio

    2009-09-01

    Methionine-choline-deficient diet represents a model for the study of the pathogenesis of steatohepatitis. Male rats were divided into three groups, the first group receiving a control diet and the other two groups receiving a methionine-choline-deficient diet for 1 month (MCD1) and for 2 months (MCD2), respectively. The livers of the animals were collected for the determination of vitamin E, thiobarbituric acid reactive substances (TBARS), GSH concentration, DNA damages, and for histopathological evaluation. The hepatic TBARS and GSH content was higher (P < 0.05) in the groups receiving the experimental diet (MCD1 and MCD2) compared to control diet, and hepatic vitamin E concentration differed (P < 0.05) between the MCD1 and MCD2 groups, with the MCD2 group presenting a lower concentration. Damage to hepatocyte DNA was greater (P < 0.05) in the MCD2 group (262.80 DNA injuries/100 hepatocytes) compared to MCD1 (136.4 DNA injuries/100 hepatocytes) and control diet (115.83 DNA injuries/100 hepatocytes). Liver histopathological evaluation showed that steatosis, present in experimental groups was micro- and macro-vesicular and concentrated around the centrolobular vein, zone 3, with preservation of the portal space. The inflammatory infiltrate was predominantly periductal and the steatosis and inflammatory infiltrate was similar in the MCD1 and MCD2 groups, although the presence of Mallory bodies was greater in the MCD2 group. The study describes the contribution of a methionine-choline-deficient diet to the progression of steatosis, lipid peroxidation and hepatic DNA damage in rats, serving as a point of reflection about the role of these nutrients in the western diet and the elevated non-alcoholic steatohepatitis rates in humans.

  9. Evidence in support of lysine 77 and histidine 96 as acid-base catalytic residues in saccharopine dehydrogenase from Saccharomyces cerevisiae.

    PubMed

    Kumar, Vidya Prasanna; Thomas, Leonard M; Bobyk, Kostyantyn D; Andi, Babak; Cook, Paul F; West, Ann H

    2012-01-31

    Saccharopine dehydrogenase (SDH) catalyzes the final reaction in the α-aminoadipate pathway, the conversion of l-saccharopine to l-lysine (Lys) and α-ketoglutarate (α-kg) using NAD⁺ as an oxidant. The enzyme utilizes a general acid-base mechanism to conduct its reaction with a base proposed to accept a proton from the secondary amine of saccharopine in the oxidation step and a group proposed to activate water to hydrolyze the resulting imine. Crystal structures of an open apo form and a closed form of the enzyme with saccharopine and NADH bound have been determined at 2.0 and 2.2 Å resolution, respectively. In the ternary complex, a significant movement of domain I relative to domain II that closes the active site cleft between the two domains and brings H96 and K77 into the proximity of the substrate binding site is observed. The hydride transfer distance is 3.6 Å, and the side chains of H96 and K77 are properly positioned to act as acid-base catalysts. Preparation of the K77M and H96Q single-mutant and K77M/H96Q double-mutant enzymes provides data consistent with their role as the general acid-base catalysts in the SDH reaction. The side chain of K77 initially accepts a proton from the ε-amine of the substrate Lys and eventually donates it to the imino nitrogen as it is reduced to a secondary amine in the hydride transfer step, and H96 protonates the carbonyl oxygen as the carbinolamine is formed. The K77M, H976Q, and K77M/H96Q mutant enzymes give 145-, 28-, and 700-fold decreases in V/E(t) and >10³-fold increases in V₂/K(Lys)E(t) and V₂/K(α-kg)E(t) (the double mutation gives >10⁵-fold decreases in the second-order rate constants). In addition, the K77M mutant enzyme exhibits a primary deuterium kinetic isotope effect of 2.0 and an inverse solvent deuterium isotope effect of 0.77 on V₂/K(Lys). A value of 2.0 was also observed for (D)(V₂/K(Lys))(D₂O) when the primary deuterium kinetic isotope effect was repeated in D₂O, consistent with a

  10. Evidence in support of lysine 77 and histidine 96 as acid-base catalytic residues in saccharopine dehydrogenase from Saccharomyces cerevisiae.

    PubMed

    Kumar, Vidya Prasanna; Thomas, Leonard M; Bobyk, Kostyantyn D; Andi, Babak; Cook, Paul F; West, Ann H

    2012-01-31

    Saccharopine dehydrogenase (SDH) catalyzes the final reaction in the α-aminoadipate pathway, the conversion of l-saccharopine to l-lysine (Lys) and α-ketoglutarate (α-kg) using NAD⁺ as an oxidant. The enzyme utilizes a general acid-base mechanism to conduct its reaction with a base proposed to accept a proton from the secondary amine of saccharopine in the oxidation step and a group proposed to activate water to hydrolyze the resulting imine. Crystal structures of an open apo form and a closed form of the enzyme with saccharopine and NADH bound have been determined at 2.0 and 2.2 Å resolution, respectively. In the ternary complex, a significant movement of domain I relative to domain II that closes the active site cleft between the two domains and brings H96 and K77 into the proximity of the substrate binding site is observed. The hydride transfer distance is 3.6 Å, and the side chains of H96 and K77 are properly positioned to act as acid-base catalysts. Preparation of the K77M and H96Q single-mutant and K77M/H96Q double-mutant enzymes provides data consistent with their role as the general acid-base catalysts in the SDH reaction. The side chain of K77 initially accepts a proton from the ε-amine of the substrate Lys and eventually donates it to the imino nitrogen as it is reduced to a secondary amine in the hydride transfer step, and H96 protonates the carbonyl oxygen as the carbinolamine is formed. The K77M, H976Q, and K77M/H96Q mutant enzymes give 145-, 28-, and 700-fold decreases in V/E(t) and >10³-fold increases in V₂/K(Lys)E(t) and V₂/K(α-kg)E(t) (the double mutation gives >10⁵-fold decreases in the second-order rate constants). In addition, the K77M mutant enzyme exhibits a primary deuterium kinetic isotope effect of 2.0 and an inverse solvent deuterium isotope effect of 0.77 on V₂/K(Lys). A value of 2.0 was also observed for (D)(V₂/K(Lys))(D₂O) when the primary deuterium kinetic isotope effect was repeated in D₂O, consistent with a

  11. Transsulfuration in an adult with hepatic methionine adenosyltransferase deficiency.

    PubMed Central

    Gahl, W A; Bernardini, I; Finkelstein, J D; Tangerman, A; Martin, J J; Blom, H J; Mullen, K D; Mudd, S H

    1988-01-01

    We investigated sulfur and methyl group metabolism in a 31-yr-old man with partial hepatic methionine adenosyltransferase (MAT) deficiency. The patient's cultured fibroblasts and erythrocytes had normal MAT activity. Hepatic S-adenosylmethionine (SAM) was slightly decreased. This clinically normal individual lives with a 20-30-fold elevation of plasma methionine (0.72 mM). He excretes in his urine methionine and L-methionine-d-sulfoxide (2.7 mmol/d), a mixed disulfide of methanethiol and a thiol bound to an unidentified group X, which we abbreviate CH3S-SX (2.1 mmol/d), and smaller quantities of 4-methylthio-2-oxobutyrate and 3-methylthiopropionate. His breath contains 17-fold normal concentrations of dimethylsulfide. He converts only 6-7 mmol/d of methionine sulfur to inorganic sulfate. This abnormally low rate is due not to a decreased flux through the primarily defective enzyme, MAT, since SAM is produced at an essentially normal rate of 18 mmol/d, but rather to a rate of homocysteine methylation which is abnormally high in the face of the very elevated methionine concentrations demonstrated in this patient. These findings support the view that SAM (which is marginally low in this patient) is an important regulator that helps to determine the partitioning of homocysteine between degradation via cystathionine and conservation by reformation of methionine. In addition, these studies demonstrate that the methionine transamination pathway operates in the presence of an elevated body load of that amino acid in human beings, but is not sufficient to maintain methionine levels in a normal range. PMID:3339126

  12. [Absorption of amino acids from the perfused ovine rumen].

    PubMed

    L' Leng; Tomás, J; Várady, J; Szányiová, M

    1978-06-01

    The experiments with extracoroporeal perfusion of sheep rumen were performed [Leng et al., 1977]. Bovine plasma, diluted in a 1:1ratio with an isotonic solution of sodium chloride, was used for four perfusions, and autologous blood was used for two perfusions in the course of 150 minutes. After 60 minutes perfusion 20 g enzymatic casein hydrolyzate were applied to the rumen. The levels of free amino acids in the perfusate were recorded after 60 minutes' perfusion [the first phase of perfusion] and at the end of the experiment [the second phase]. The levels of lysine, aspartic acid and glutamic acid increased after perfusions with bovine plasma during the first phase, the levels of glutamic acid, phenylalanine, and in one case of alanine, increased after perfusions with autologus blood. Simultaneously the level of valine decreased after perfusions with bovine plasma, and after perfusions with blood the levels of arginine and valine, and/or lysine, dropped. During the second phase of perfusion, the levels of all the observed amino acids except methionine [bovine plasma], and/or orginine and methionine [blood] rose in the perfusate. The experiments showed that the level of amino acids in the rumen content presented a decisive factor affecting amino acid absorption from the rumen into the blood. Transformation of the amino acids during their passage through the remen wall may be assumed, and glutamic acid is one of the chief products of this process.

  13. Potential for Development of an Escherichia coli—Based Biosensor for Assessing Bioavailable Methionine: A Review

    PubMed Central

    Chalova, Vesela I.; Froelich, Clifford A.; Ricke, Steven C.

    2010-01-01

    Methionine is an essential amino acid for animals and is typically considered one of the first limiting amino acids in animal feed formulations. Methionine deficiency or excess in animal diets can lead to sub-optimal animal performance and increased environmental pollution, which necessitates its accurate quantification and proper dosage in animal rations. Animal bioassays are the current industry standard to quantify methionine bioavailability. However, animal-based assays are not only time consuming, but expensive and are becoming more scrutinized by governmental regulations. In addition, a variety of artifacts can hinder the variability and time efficacy of these assays. Microbiological assays, which are based on a microbial response to external supplementation of a particular nutrient such as methionine, appear to be attractive potential alternatives to the already established standards. They are rapid and inexpensive in vitro assays which are characterized with relatively accurate and consistent estimation of digestible methionine in feeds and feed ingredients. The current review discusses the potential to develop Escherichia coli-based microbial biosensors for methionine bioavailability quantification. Methionine biosynthesis and regulation pathways are overviewed in relation to genetic manipulation required for the generation of a respective methionine auxotroph that could be practical for a routine bioassay. A prospective utilization of Escherichia coli methionine biosensor would allow for inexpensive and rapid methionine quantification and ultimately enable timely assessment of nutritional profiles of feedstuffs. PMID:22319312

  14. Oxidation of methionine residues in polypeptide ions via gas-phase ion/ion chemistry.

    PubMed

    Pilo, Alice L; McLuckey, Scott A

    2014-06-01

    The gas-phase oxidation of methionine residues is demonstrated here using ion/ion reactions with periodate anions. Periodate anions are observed to attach in varying degrees to all polypeptide ions irrespective of amino acid composition. Direct proton transfer yielding a charge-reduced peptide ion is also observed. In the case of methionine and, to a much lesser degree, tryptophan-containing peptide ions, collisional activation of the complex ion generated by periodate attachment yields an oxidized peptide product (i.e., [M + H + O](+)), in addition to periodic acid detachment. Detachment of periodic acid takes place exclusively for peptides that do not contain either a methionine or tryptophan side chain. In the case of methionine-containing peptides, the [M + H + O](+) product is observed at a much greater abundance than the proton transfer product (viz., [M + H](+)). Collisional activation of oxidized Met-containing peptides yields a signature loss of 64 Da from the precursor and/or product ions. This unique loss corresponds to the ejection of methanesulfenic acid from the oxidized methionine side chain and is commonly used in solution-phase proteomics studies to determine the presence of oxidized methionine residues. The present work shows that periodate anions can be used to 'label' methionine residues in polypeptides in the gas phase. The selectivity of the periodate anion for the methionine side chain suggests several applications including identification and location of methionine residues in sequencing applications.

  15. Oxidation of Methionine Residues in Polypeptide Ions Via Gas-Phase Ion/Ion Chemistry

    NASA Astrophysics Data System (ADS)

    Pilo, Alice L.; McLuckey, Scott A.

    2014-06-01

    The gas-phase oxidation of methionine residues is demonstrated here using ion/ion reactions with periodate anions. Periodate anions are observed to attach in varying degrees to all polypeptide ions irrespective of amino acid composition. Direct proton transfer yielding a charge-reduced peptide ion is also observed. In the case of methionine and, to a much lesser degree, tryptophan-containing peptide ions, collisional activation of the complex ion generated by periodate attachment yields an oxidized peptide product (i.e., [M + H + O]+), in addition to periodic acid detachment. Detachment of periodic acid takes place exclusively for peptides that do not contain either a methionine or tryptophan side chain. In the case of methionine-containing peptides, the [M + H + O]+ product is observed at a much greater abundance than the proton transfer product (viz., [M + H]+). Collisional activation of oxidized Met-containing peptides yields a signature loss of 64 Da from the precursor and/or product ions. This unique loss corresponds to the ejection of methanesulfenic acid from the oxidized methionine side chain and is commonly used in solution-phase proteomics studies to determine the presence of oxidized methionine residues. The present work shows that periodate anions can be used to `label' methionine residues in polypeptides in the gas phase. The selectivity of the periodate anion for the methionine side chain suggests several applications including identification and location of methionine residues in sequencing applications.

  16. Autologous aldrithiol-2-inactivated HIV-1 combined with polyinosinic-polycytidylic acid-poly-L-lysine carboxymethylcellulose as a vaccine platform for therapeutic dendritic cell immunotherapy.

    PubMed

    Miller, Elizabeth; Spadaccia, Meredith; Sabado, Rachel; Chertova, Elena; Bess, Julian; Trubey, Charles Mac; Holman, Rose Marie; Salazar, Andres; Lifson, Jeffrey; Bhardwaj, Nina

    2015-01-01

    Therapeutic interventions for HIV-1 that successfully augment adaptive immunity to promote killing of infected cells may be a requisite component of strategies to reduce latent cellular reservoirs. Adoptive immunotherapies utilizing autologous monocyte-derived dendritic cells (DCs) that have been activated and antigen loaded ex vivo may serve to circumvent defects in DC function that are present during HIV infection in order to enhance adaptive immune responses. Here we detail the clinical preparation of DCs loaded with autologous aldrithiol-2 (AT-2)-inactivated HIV that have been potently activated with the viral mimic, Polyinosinic-polycytidylic acid-poly-l-lysine carboxymethylcellulose (Poly-ICLC). HIV is first propagated from CD4+ T cells from HIV-infected donors and then rendered non-replicative by chemical inactivation with aldrithiol-2 (AT-2), purified, and quantified. Viral inactivation is confirmed through measurement of Tat-regulated β-galactosidase reporter gene expression following infection of TZM-bl cells. In-process testing for sterility, mycoplasma, LPS, adventitious agents, and removal of AT-2 is performed on viral preparations. Autologous DCs are generated and pulsed with autologous AT-2-inactivated virus and simultaneously stimulated with Poly-ICLC to constitute the final DC vaccine product. Phenotypic identity, maturation, and induction of HIV-specific adaptive immune responses are confirmed via flow cytometric analysis of DCs and cocultured autologous CD4+ and CD8+ T cells. Lot release criteria for the DC vaccine have been defined in accordance with Good Manufacturing Practice (GMP) guidelines. The demonstrated feasibility of this approach has resulted in approval by the FDA for investigational use in antiretroviral (ART) suppressed individuals. We discuss how this optimized DC formulation may enhance the quality of anti-HIV adaptive responses beyond what has been previously observed during DC immunotherapy trials for HIV infection.

  17. Effect of L-lysine on expression of selected genes, serum concentration of amino acids, muscle growth and performance of growing pigs.

    PubMed

    Morales, A; García, H; Arce, N; Cota, M; Zijlstra, R T; Araiza, B A; Cervantes, M

    2015-08-01

    Lysine (Lys) is the first limiting amino acid (AA) in most feed formulations for pigs and most abundant, along with leucine, in muscle proteins. An experiment was conducted with 17 pigs (17.7 ± 0.05 kg initial BW) to identify a role of dietary Lys in the control of protein synthesis in pigs. Fourteen pigs were randomly assigned to one of the two wheat-based dietary treatments: Lys-deficient, 3.0 g/kg (DEF) and Lys-adequate, 10.8 g/kg (ADE). Samples from jejunum mucosa, liver, Longissumus and Semitendinosus muscles, and blood were collected. The other three pigs were sacrificed at the beginning of the trial to measure basal carcass composition. Weight gain, gain:feed ratio, Lys intake and loin eye area were greater in ADE than in DEF pigs (p < 0.01). Muscle-related carcass characteristics were better, and myosin heavy chain IIb expression (MyHC IIb) in Semitendinosus was higher in ADE than in DEF pigs. Expression of AA transporters CAT-1 was lower (p < 0.05), serum Lys was higher and serum Val was lower in pigs fed the ADE diet. The higher muscularity, MyHC IIb expression in Semitendinosus muscle and Lys serum of pigs fed the ADE diet suggest that Lys increases growth rate not only by functioning as protein construction unit but also as potential control of the protein synthesis process.

  18. Methionine salvage pathway in relation to ethylene biosynthesis

    SciTech Connect

    Miyazaki, J.H.

    1987-01-01

    The recycling of methionine during ethylene biosynthesis (the methionine cycle) was studied. During ethylene biosynthesis, the H/sub 3/CS-group of S-adenosylmethionine (SAM) is released at 5'-methylthioadenosine (MTA), which is recycled to methionine via 5'-methylthioribose (MTS). In mungbean hypocotyls and cell-free extracts of avocado fruit, (/sup 14/C)MTR was converted to labeled methionine via 2-keto-4-methylthiobutyric acid (KMB) and 2-hydroxy-4-methylthiobutyric acid (HMB) as intermediates. Radioactive tracer studies showed that KMB was converted readily in vivo and in vitro to methionine, while HMB was converted much more slowly. The conversion of KMB to methionine by dialyzed avocado extract required an amino group donor. Among several potential donors tested, L-glutamine was the most efficient. Incubation of (ribose-U-/sup 14/C)MTR with avocado extract resulted in the production of (/sup 14/C)formate, with little evolution of other /sup 14/C-labeled one-carbon compounds, indicating that the conversion of MTR to KMB involves a loss of formate, presumably from C-1 of MTR.

  19. Thyroid metabolism in the recessive sex-linked dwarf female chicken. 4. The influence of exogenous thyroid hormones on amino acid uptake by plasma and tissues.

    PubMed

    Grandhi, R R; Brown, R G; Reinhart, B S; Summers, J D

    1975-03-01

    The influence of exogenous triiodothyronine (T3) or tetraiodothyronine (T4) on the incorporation of 3H-labelled methionine, alanine and lysine into plasma, liver and kidney was studied in 4 wk. old dwarf and non-dwarf female, White Leghorn chickens. The response to exogenous T3 or T4 of the birds was directly dependent on the dwarf status as well as the tissue and/or amino acid studied. In general, there was a decreased amino acid uptake by dwarfs and T3 and/or T4 depressed amino acid uptake in all combinations studied except for the uptake of lysine by the kidney of the dwarf. In that tissue, T4 administration caused a significant increase in lysine incorporation. The results found probably were due to different tissue amino compositions or rates of synthesis of proteins by dwarf birds when compared to normals and a differential sensitivity of dwarf birds to changed T3/T4 ratios.

  20. Selenium and Methionine Sulfoxide Reduction.

    PubMed

    Gladyshev, Vadim N

    2014-10-01

    Selenium is an essential trace element because it is present in proteins in the form of selenocysteine residue. Functionally characterized selenoproteins are oxidoreductases. Selenoprotein methionine-R-sulfoxide reductase B1 (MsrB1) is a repair enzyme that reduces ROS-oxidized methionine residues in proteins. Here, we explored a possibility that reversible methionine oxidation is also a mechanism that regulates protein function. We found that MsrB1, together with Mical proteins, regulated mammalian actin assembly via stereospecific methionine oxidation and reduction in a reversible, site-specific manner. Two methionine residues in actin were specifically converted to methionine-R-sulfoxide by Mical1 and Mical2 and reduced back to methionine by MsrB1, supporting actin disassembly and assembly, respectively. Macrophages utilized this redox control during cellular activation by stimulating MsrB1 expression and activity. Thus, we identified the regulatory role of MsrB1 as a Mical antagonist in orchestrating actin dynamics and macrophage function. More generally, our study showed that proteins can be regulated by reversible site-specific methionine-R-sulfoxidation and that selenium is involved in this regulation by being a catalytic component of MsrB1. PMID:26461418

  1. Interrelationship between methionine and cystine of early Peking ducklings.

    PubMed

    Xie, M; Hou, S S; Huang, W; Zhao, L; Yu, J Y; Li, W Y; Wu, Y Y

    2004-10-01

    A 4 x 5 factorial experiment containing 4 cystine levels (0.325, 0.406, 0.487, or 0.568%) and 5 methionine levels (0.285, 0.385, 0.485, 0.585, or 0.685%) was conducted to evaluate the interrelationship between methionine and cystine in corn-peanut meal diet for Peking ducklings from hatch to 21 d of age. Eight hundred 1-d-old male white Peking ducklings were assigned to 20 experimental treatments. All treatments were replicated 4 times using 10 ducklings per pen. As dietary methionine level increased, weight gain and feed intake increased and then decreased; the quadratic response of weight gain was significant (P < 0.05). The methionine requirement for maximum efficiency of feed utilization (0.585%) was higher than for maximum weight gain (0.485%). According to the quadratic model, the optimal methionine requirement of Peking ducklings from hatch to 21 d of age was 0.481% (95% of the level at maximum response). The plasma uric acid concentration was very low (P < 0.05) when dietary methionine was 0.485%. When dietary methionine was excessive (0.685%), the plasma homocysteine concentration increased (P < 0.05). On the other hand, the cystine requirement of ducklings from hatch to 21 d of age was not more than 0.325%. A high level of cystine (0.568%) depressed weight gain and feed intake (P < 0.05), but cystine supplementation in the diets lowered the plasma homocysteine concentration (P < 0.05). There were no significant interactions between methionine and cystine on growth performance, plasma uric acid, and plasma homocysteine.

  2. Accumulation, selection and covariation of amino acids in sieve tube sap of tansy (Tanacetum vulgare) and castor bean (Ricinus communis): evidence for the function of a basic amino acid transporter and the absence of a γ-amino butyric acid transporter.

    PubMed

    Bauer, Susanne N; Nowak, Heike; Keller, Frank; Kallarackal, Jose; Hajirezaei, Mohamad-Reza; Komor, Ewald

    2014-09-01

    Sieve tube sap was obtained from Tanacetum by aphid stylectomy and from Ricinus after apical bud decapitation. The amino acids in sieve tube sap were analyzed and compared with those from leaves. Arginine and lysine accumulated in the sieve tube sap of Tanacetum more than 10-fold compared to the leaf extracts and they were, together with asparagine and serine, preferably selected into the sieve tube sap, whereas glycine, methionine/tryptophan and γ-amino butyric acid were partially or completely excluded. The two basic amino acids also showed a close covariation in sieve tube sap. The acidic amino acids also grouped together, but antagonistic to the other amino acids. The accumulation ratios between sieve tube sap and leaf extracts were smaller in Ricinus than in Tanacetum. Arginine, histidine, lysine and glutamine were enriched and preferentially loaded into the phloem, together with isoleucine and valine. In contrast, glycine and methionine/tryptophan were partially and γ-amino butyric acid almost completely excluded from sieve tube sap. The covariation analysis grouped arginine together with several neutral amino acids. The acidic amino acids were loaded under competition with neutral amino acids. It is concluded from comparison with the substrate specificities of already characterized plant amino acid transporters, that an AtCAT1-like transporter functions in phloem loading of basic amino acids, whereas a transporter like AtGAT1 is absent in phloem. Although Tanacetum and Ricinus have different minor vein architecture, their phloem loading specificities for amino acids are relatively similar.

  3. Synthesis of Lysine Methyltransferase Inhibitors

    NASA Astrophysics Data System (ADS)

    Ye, Tao; Hui, Chunngai

    2015-07-01

    Lysine methyltransferase which catalyze methylation of histone and nonhistone proteins, play a crucial role in diverse biological processes and has emerged as a promising target for the development of various human diseases, including cancer, inflammation, and psychiatric disorders. However, inhibiting Lysine methyltransferases selectively has presented many challenges to medicinal chemists. During the past decade, lysine methyltransferase inhibitors covering many different structural classes have been designed and developed. In this review, we describe the development of selective, small-molecule inhibitors of lysine methyltransferases with an emphasis on their discovery and chemical synthesis. We highlight the current state of lysine methyltransferase inhibitors and discuss future directions and opportunities for lysine methyltransferase inhibitor discovery.

  4. Protein Methionine Sulfoxide Dynamics in Arabidopsis thaliana under Oxidative Stress.

    PubMed

    Jacques, Silke; Ghesquière, Bart; De Bock, Pieter-Jan; Demol, Hans; Wahni, Khadija; Willems, Patrick; Messens, Joris; Van Breusegem, Frank; Gevaert, Kris

    2015-05-01

    Reactive oxygen species such as hydrogen peroxide can modify proteins via direct oxidation of their sulfur-containing amino acids, cysteine and methionine. Methionine oxidation, studied here, is a reversible posttranslational modification that is emerging as a mechanism by which proteins perceive oxidative stress and function in redox signaling. Identification of proteins with oxidized methionines is the first prerequisite toward understanding the functional effect of methionine oxidation on proteins and the biological processes in which they are involved. Here, we describe a proteome-wide study of in vivo protein-bound methionine oxidation in plants upon oxidative stress using Arabidopsis thaliana catalase 2 knock-out plants as a model system. We identified over 500 sites of oxidation in about 400 proteins and quantified the differences in oxidation between wild-type and catalase 2 knock-out plants. We show that the activity of two plant-specific glutathione S-transferases, GSTF9 and GSTT23, is significantly reduced upon oxidation. And, by sampling over time, we mapped the dynamics of methionine oxidation and gained new insights into this complex and dynamic landscape of a part of the plant proteome that is sculpted by oxidative stress.

  5. Reevaluation of the rate constants for the reaction of hypochlorous acid (HOCl) with cysteine, methionine, and peptide derivatives using a new competition kinetic approach.

    PubMed

    Storkey, Corin; Davies, Michael J; Pattison, David I

    2014-08-01

    Activated white cells use oxidants generated by the heme enzyme myeloperoxidase to kill invading pathogens. This enzyme utilizes H2O2 and Cl(-), Br(-), or SCN(-) to generate the oxidants HOCl, HOBr, and HOSCN, respectively. Whereas controlled production of these species is vital in maintaining good health, their uncontrolled or inappropriate formation (as occurs at sites of inflammation) can cause host tissue damage that has been associated with multiple inflammatory pathologies including cardiovascular diseases and cancer. Previous studies have reported that sulfur-containing species are major targets for HOCl but as the reactions are fast the only physiologically relevant kinetic data available have been extrapolated from data measured at high pH (>10). In this study these values have been determined at pH 7.4 using a newly developed competition kinetic approach that employs a fluorescently tagged methionine derivative as the competitive substrate (k(HOCl + Fmoc-Met), 1.5 × 10(8)M(-1)s(-1)). This assay was validated using the known k(HOCl + NADH) value and has allowed revised k values for the reactions of HOCl with Cys, N-acetylcysteine, and glutathione to be determined as 3.6 × 10(8), 2.9 × 10(7), and 1.24 × 10(8)M(-1)s(-1), respectively. Similar experiments with methionine derivatives yielded k values of 3.4 × 10(7)M(-1)s(-1) for Met and 1.7 × 10(8)M(-1)s(-1) for N-acetylmethionine. The k values determined here for the reaction of HOCl with thiols are up to 10-fold higher than those previously determined and further emphasize the critical importance of reactions of HOCl with thiol targets in biological systems.

  6. Comparative evaluation of transport mechanisms of trans-1-amino-3-[¹⁸F]fluorocyclobutanecarboxylic acid and L-[methyl-¹¹C]methionine in human glioma cell lines.

    PubMed

    Ono, Masahiro; Oka, Shuntaro; Okudaira, Hiroyuki; Schuster, David M; Goodman, Mark M; Kawai, Keiichi; Shirakami, Yoshifumi

    2013-10-16

    Positron emission tomography (PET) with amino acid tracers is useful for the visualization and assessment of therapeutic effects on gliomas. Our purpose is to elucidate the transport mechanisms of trans-1-amino-3-[¹⁸F]fluorocyclobutanecarboxylic acid (anti-[¹⁸F]FACBC) and L-[methyl-¹¹C]methionine ([¹¹C]Met) in normal human astrocytes (NHA), low-grade (Hs683, SW1088), and high-grade (U87MG, T98G) human glioma cell lines. Because the short half-lives of fluorine-18 and carbon-11 are inconvenient for in vitro experiments, trans-1-amino-3-fluoro[1-¹⁴C]cyclobutanecarboxylic acid (anti-[¹⁴C]FACBC) and L-[methyl-¹⁴C]methionine ([¹⁴C]Met) were used instead of the PET tracers. Time-course uptake experiments showed that uptake of anti-[¹⁴C]FACBC was 1.4-2.6 times higher than that of [¹⁴C]Met in NHA and low-grade glioma cells, and was almost equal to that of [¹⁴C]Met in high-grade glioma cells. To identify the amino acid transporters (AATs) involved in the transport of anti-[¹⁴C]FACBC and [¹⁴C]Met, we carried out competitive inhibition experiments using synthetic/naturally-occurring amino acids as inhibitors. We found that anti-[¹⁴C]FACBC uptake in the presence of Na⁺ was strongly inhibited by L-glutamine and L-serine (the substrates for ASC system AATs), whereas L-phenylalanine and 2-amino-bicyclo[2,2,1]heptane-2-carboxylic acid (BCH, the substrates for L system AATs) robustly inhibited Na⁺-independent anti-[¹⁴C]FACBC uptake. Regardless of Na⁺, [¹⁴C]Met uptake was inhibited strongly by L-phenylalanine and BCH. Moreover, the exchange transport activity of L-glutamine for anti-[¹⁴C]FACBC was stronger than that of BCH in the presence of Na⁺, whereas that for [¹⁴C]Met was almost equal to BCH. These results demonstrate that ASC and L are important transport systems for anti-[¹⁸F]FACBC uptake, while system L is predominantly involved in [¹¹C]Met transport in human astrocytes and glioma cells.

  7. The low-methionine content of vegan diets may make methionine restriction feasible as a life extension strategy.

    PubMed

    McCarty, Mark F; Barroso-Aranda, Jorge; Contreras, Francisco

    2009-02-01

    Recent studies confirm that dietary methionine restriction increases both mean and maximal lifespan in rats and mice, achieving "aging retardant" effects very similar to those of caloric restriction, including a suppression of mitochondrial superoxide generation. Although voluntary caloric restriction is never likely to gain much popularity as a pro-longevity strategy for humans, it may be more feasible to achieve moderate methionine restriction, in light of the fact that vegan diets tend to be relatively low in this amino acid. Plant proteins - especially those derived from legumes or nuts - tend to be lower in methionine than animal proteins. Furthermore, the total protein content of vegan diets, as a function of calorie content, tends to be lower than that of omnivore diets, and plant protein has somewhat lower bioavailability than animal protein. Whole-food vegan diets that moderate bean and soy intake, while including ample amounts of fruit and wine or beer, can be quite low in methionine, while supplying abundant nutrition for health (assuming concurrent B12 supplementation). Furthermore, low-fat vegan diets, coupled with exercise training, can be expected to promote longevity by decreasing systemic levels of insulin and free IGF-I; the latter effect would be amplified by methionine restriction - though it is not clear whether IGF-I down-regulation is the sole basis for the impact of low-methionine diets on longevity in rodents.

  8. Serine hydroxymethyltransferase: a key player connecting purine, folate and methionine metabolism in Saccharomyces cerevisiae.

    PubMed

    Saint-Marc, Christelle; Hürlimann, Hans C; Daignan-Fornier, Bertrand; Pinson, Benoît

    2015-11-01

    Previous genetic analyses showed phenotypic interactions between 5-amino-4-imidazole carboxamide ribonucleotide 5'-phosphate (AICAR) produced from the purine and histidine pathways and methionine biosynthesis. Here, we revisited the effect of AICAR on methionine requirement due to AICAR accumulation in the presence of the fau1 mutation invalidating folinic acid remobilization. We found that this methionine auxotrophy could be suppressed by overexpression of the methionine synthase Met6 or by deletion of the serine hydroxymethyltransferase gene SHM2. We propose that in a fau1 background, AICAR, by stimulating the transcriptional expression of SHM2, leads to a folinic acid accumulation inhibiting methionine synthesis by Met6. In addition, we uncovered a new methionine auxotrophy for the ade3 bas1 double mutant that can be rescued by overexpressing the SHM2 gene. We propose that methionine auxotrophy in this mutant is the result of a competition for 5,10-methylenetetrahydrofolate between methionine and deoxythymidine monophosphate synthesis. Altogether, our data show intricate genetic interactions between one-carbon units, purine and methionine metabolism through fine-tuning of serine hydroxymethyltransferase by AICAR and the transcription factor Bas1.

  9. l-Methionine Placental Uptake

    PubMed Central

    Araújo, João R.; Correia-Branco, Ana; Ramalho, Carla; Gonçalves, Pedro; Pinho, Maria J.; Keating, Elisa

    2013-01-01

    Our aim was to investigate the influence of gestational diabetes mellitus (GDM) and GDM-associated conditions upon the placental uptake of 14C-l-methionine (14C-l-Met). The 14C-l-Met uptake by human trophoblasts (TBs) obtained from normal pregnancies (normal trophoblast [NTB] cells) is mainly system l-type amino acid transporter 1 (LAT1 [L])-mediated, although a small contribution of system y+LAT2 is also present. Comparison of 14C-l-Met uptake by NTB and by human TBs obtained from GDM pregnancies (diabetic trophoblast [DTB] cells) reveals similar kinetics, but a contribution of systems A, LAT2, and b0+ and a greater contribution of system y+LAT1 appears to exist in DTB cells. Short-term exposure to insulin and long-term exposure to high glucose, tumor necrosis factor-α, and leptin decrease 14C-l-Met uptake in a human TB (Bewo) cell line. The effect of leptin was dependent upon phosphoinositide 3-kinase, extracellular-signal-regulated kinase 1/2 (ERK/MEK 1/2), and p38 mitogen-activated protein kinase. In conclusion, GDM does not quantitatively alter 14C-l-Met placental uptake, although it changes the nature of transporters involved in that process. PMID:23653387

  10. Disruption of Methionine Metabolism in Drosophila melanogaster Impacts Histone Methylation and Results in Loss of Viability

    PubMed Central

    Liu, Mengying; Barnes, Valerie L.; Pile, Lori A.

    2015-01-01

    Histone methylation levels, which are determined by the action of both histone demethylases and methyltransferases, impact multiple biological processes by affecting gene expression activity. Methionine metabolism generates the major methyl donor S-adenosylmethionine (SAM) for histone methylation. The functions of methionine metabolic enzymes in regulating biological processes as well as the interaction between the methionine pathway and histone methylation, however, are still not fully understood. Here, we report that reduced levels of some enzymes involved in methionine metabolism and histone demethylases lead to lethality as well as wing development and cell proliferation defects in Drosophila melanogaster. Additionally, disruption of methionine metabolism can directly affect histone methylation levels. Reduction of little imaginal discs (LID) histone demethylase, but not lysine-specific demethylase 2 (KDM2) demethylase, is able to counter the effects on histone methylation due to reduction of SAM synthetase (SAM-S). Taken together, these results reveal an essential role of key enzymes that control methionine metabolism and histone methylation. Additionally, these findings are an indication of a strong connection between metabolism and epigenetics. PMID:26546310

  11. Effect of excess methionine and methionine hydroxy analogue on growth performance and plasma homocysteine of growing Pekin ducks.

    PubMed

    Xie, M; Hou, S S; Huang, W; Fan, H P

    2007-09-01

    One experiment was conducted to study the effect of excess dl-methionine (DLM) and dl-2-hydroxy-4-methylthiobutanoic acid free acid (dl-HMB-FA) on duck growth. One-day-old male white Pekin ducklings were fed common starter diets from hatch to 21 d of age and then fed the experimental diets from 21 to 42 d of age. Three hundred twenty 21-d-old birds were allotted to 40 raised wire-floor pens with 8 birds per pen according to similar pen weight. There were 5 dietary treatments that included a methionine-adequate control diet and control diets supplemented with 2 levels of dry DLM (1 or 2%) or 2 equimolar levels of liquid dl-HMB-FA (1.13 or 2.26%). Each dietary treatment was replicated 8 times. At 42 d of age, weight gain, feed intake, and gain/feed were measured and plasma was collected to analyze homocysteine. Compared with ducks fed control diets, excess DLM or dl-HMB-FA supplementation reduced weight gain and feed intake of birds significantly. However, on the equimolar basis, at 1 or 2% supplemental methionine activity, dl-HMB-FA was less growth-depressing than DLM. According to the growth response to excess methionine, the tolerable upper limit of dietary methionine for growing ducks may be less than 1.38% when the methionine level of the control diet (0.38%) was considered. On the other hand, plasma homocysteine was elevated markedly when 2% DLM or 2.26% dl-HMB-FA was added to control diets, but plasma homocysteine of ducks fed 2.26% dl-HMB-FA supplemented diets was lower significantly than birds fed equimolar DLM-supplemented diets, which indicated the toxicity of excess methionine sources and less toxicity of dl-HMB-FA relative to DLM.

  12. Serine Metabolism Supports the Methionine Cycle and DNA/RNA Methylation through De Novo ATP Synthesis in Cancer Cells

    PubMed Central

    Maddocks, Oliver D.K.; Labuschagne, Christiaan F.; Adams, Peter D.; Vousden, Karen H.

    2016-01-01

    Summary Crosstalk between cellular metabolism and the epigenome regulates epigenetic and metabolic homeostasis and normal cell behavior. Changes in cancer cell metabolism can directly impact epigenetic regulation and promote transformation. Here we analyzed the contribution of methionine and serine metabolism to methylation of DNA and RNA. Serine can contribute to this pathway by providing one-carbon units to regenerate methionine from homocysteine. While we observed this contribution under methionine-depleted conditions, unexpectedly, we found that serine supported the methionine cycle in the presence and absence of methionine through de novo ATP synthesis. Serine starvation increased the methionine/S-adenosyl methionine ratio, decreasing the transfer of methyl groups to DNA and RNA. While serine starvation dramatically decreased ATP levels, this was accompanied by lower AMP and did not activate AMPK. This work highlights the difference between ATP turnover and new ATP synthesis and defines a vital function of nucleotide synthesis beyond making nucleic acids. PMID:26774282

  13. Serine Metabolism Supports the Methionine Cycle and DNA/RNA Methylation through De Novo ATP Synthesis in Cancer Cells.

    PubMed

    Maddocks, Oliver D K; Labuschagne, Christiaan F; Adams, Peter D; Vousden, Karen H

    2016-01-21

    Crosstalk between cellular metabolism and the epigenome regulates epigenetic and metabolic homeostasis and normal cell behavior. Changes in cancer cell metabolism can directly impact epigenetic regulation and promote transformation. Here we analyzed the contribution of methionine and serine metabolism to methylation of DNA and RNA. Serine can contribute to this pathway by providing one-carbon units to regenerate methionine from homocysteine. While we observed this contribution under methionine-depleted conditions, unexpectedly, we found that serine supported the methionine cycle in the presence and absence of methionine through de novo ATP synthesis. Serine starvation increased the methionine/S-adenosyl methionine ratio, decreasing the transfer of methyl groups to DNA and RNA. While serine starvation dramatically decreased ATP levels, this was accompanied by lower AMP and did not activate AMPK. This work highlights the difference between ATP turnover and new ATP synthesis and defines a vital function of nucleotide synthesis beyond making nucleic acids.

  14. Methionine restriction inhibits chemically-induced malignant transformation in the BALB/c 3T3 cell transformation assay.

    PubMed

    Nicken, Petra; Empl, Michael T; Gerhard, Daniel; Hausmann, Julia; Steinberg, Pablo

    2016-09-01

    High consumption of red meat entails a higher risk of developing colorectal cancer. Methionine, which is more frequently a component of animal proteins, and folic acid are members of the one carbon cycle and as such important players in DNA methylation and cancer development. Therefore, dietary modifications involving altered methionine and folic acid content might inhibit colon cancer development. In the present study, the BALB/c 3T3 cell transformation assay was used to investigate whether methionine and folic acid are able to influence the malignant transformation of mouse fibroblasts after treatment with the known tumour initiator 3-methylcholanthrene. Three different methionine concentrations (representing a -40%, a "normal" and a +40% cell culture medium concentration, respectively) and two different folic acid concentrations (6 and 20 μM) were thereby investigated. Methionine restriction led to a decrease of type III foci, while enhancement of both methionine and folic acid did not significantly increase the cell transformation rate. Interestingly, the focus-lowering effect of methionine was only significant in conjunction with an elevated folic acid concentration. In summary, we conclude that the malignant transformation of mouse fibroblasts is influenced by methionine levels and that methionine restriction could be a possible approach to reduce cancer development. PMID:27427305

  15. Methionine restriction inhibits chemically-induced malignant transformation in the BALB/c 3T3 cell transformation assay.

    PubMed

    Nicken, Petra; Empl, Michael T; Gerhard, Daniel; Hausmann, Julia; Steinberg, Pablo

    2016-09-01

    High consumption of red meat entails a higher risk of developing colorectal cancer. Methionine, which is more frequently a component of animal proteins, and folic acid are members of the one carbon cycle and as such important players in DNA methylation and cancer development. Therefore, dietary modifications involving altered methionine and folic acid content might inhibit colon cancer development. In the present study, the BALB/c 3T3 cell transformation assay was used to investigate whether methionine and folic acid are able to influence the malignant transformation of mouse fibroblasts after treatment with the known tumour initiator 3-methylcholanthrene. Three different methionine concentrations (representing a -40%, a "normal" and a +40% cell culture medium concentration, respectively) and two different folic acid concentrations (6 and 20 μM) were thereby investigated. Methionine restriction led to a decrease of type III foci, while enhancement of both methionine and folic acid did not significantly increase the cell transformation rate. Interestingly, the focus-lowering effect of methionine was only significant in conjunction with an elevated folic acid concentration. In summary, we conclude that the malignant transformation of mouse fibroblasts is influenced by methionine levels and that methionine restriction could be a possible approach to reduce cancer development.

  16. Methionine Metabolism Alters Oxidative Stress Resistance via the Pentose Phosphate Pathway.

    PubMed

    Campbell, Kate; Vowinckel, Jakob; Keller, Markus A; Ralser, Markus

    2016-04-01

    Nutrient uptake and metabolism have a significant impact on the way cells respond to stress. The amino acid methionine is, in particular, a key player in the oxidative stress response, and acting as a reactive oxygen species scavenger, methionine is implicated in caloric restriction phenotypes and aging. We here provide evidence that some effects of methionine in stress situations are indirect and caused by altered activity of the nicotinamide adenine dinucleotide phosphate (NADPH) producing oxidative part of the pentose phosphate pathway (PPP). In Saccharomyces cerevisiae, both methionine prototrophic (MET15) and auxotrophic (met15Δ) cells supplemented with methionine showed an increase in PPP metabolite concentrations downstream of the NADPH producing enzyme, 6-phosphogluconate dehydrogenase. Proteomics revealed this enzyme to also increase in expression compared to methionine self-synthesizing cells. Oxidant tolerance was increased in cells preincubated with methionine; however, this effect was abolished when flux through the oxidative PPP was prevented by deletion of its rate limiting enzyme, ZWF1. Stress resistance phenotypes that follow methionine supplementation hence involve the oxidative PPP. Effects of methionine on oxidative metabolism, stress signaling, and aging have thus to be seen in the context of an altered activity of this NADP reducing pathway.

  17. Methionine Metabolism Alters Oxidative Stress Resistance via the Pentose Phosphate Pathway

    PubMed Central

    Campbell, Kate; Vowinckel, Jakob; Keller, Markus A.

    2016-01-01

    Abstract Nutrient uptake and metabolism have a significant impact on the way cells respond to stress. The amino acid methionine is, in particular, a key player in the oxidative stress response, and acting as a reactive oxygen species scavenger, methionine is implicated in caloric restriction phenotypes and aging. We here provide evidence that some effects of methionine in stress situations are indirect and caused by altered activity of the nicotinamide adenine dinucleotide phosphate (NADPH) producing oxidative part of the pentose phosphate pathway (PPP). In Saccharomyces cerevisiae, both methionine prototrophic (MET15) and auxotrophic (met15Δ) cells supplemented with methionine showed an increase in PPP metabolite concentrations downstream of the NADPH producing enzyme, 6-phosphogluconate dehydrogenase. Proteomics revealed this enzyme to also increase in expression compared to methionine self-synthesizing cells. Oxidant tolerance was increased in cells preincubated with methionine; however, this effect was abolished when flux through the oxidative PPP was prevented by deletion of its rate limiting enzyme, ZWF1. Stress resistance phenotypes that follow methionine supplementation hence involve the oxidative PPP. Effects of methionine on oxidative metabolism, stress signaling, and aging have thus to be seen in the context of an altered activity of this NADP reducing pathway. Antioxid. Redox Signal. 24, 543–547. PMID:26596469

  18. [Comparison of the digestive utilization of methionine, of its hydroxylated analog, and of sodium sulfate in goats using 35s compounds].

    PubMed

    Champredon, C; Pion, R; Basson, W D

    1976-02-23

    35S and 35S free and protein bound amino acids were estimated in goats' abomasal contents and blood after ruminal injections of sulfer labelled compounds: methionine, methionine hydroxy analog (M.H.A.) and sodium sulfate. 35S incorporation into microbial and plasma proteins was higher with methionine than with M.H.A. or sulfate. 35S.M.H.A. utilisation seems to be less different from Na2 35SO4 utilisation than from 35S methionine utilisation.

  19. Influence of dietary methionine on the metabolism of selenomethionine in rats

    SciTech Connect

    Butler, J.A.; Beilstein, M.A.; Whanger, P.D. )

    1989-07-01

    To determine the influence of methionine on selenomethionine (SeMet) metabolism, weanling male rats were fed for 8 wk a basal diet marginally deficient in sulfur amino acids, containing 2.0 micrograms selenium (Se)/g as DL-SeMet and supplemented with 0, 0.3, 0.6 or 1.2% DL-methionine. Increased dietary methionine caused decreased selenium deposition in all tissues examined but increased glutathione peroxidase activity in testes, liver and lungs. A positive correlation was found between dietary methionine and the calculated percentage of selenium associated with GSHPx. In a second experiment, {sup 75}SeMet was injected into weanling male rats which had been fed the basal diet containing 2.0 micrograms selenium as DL-SeMet with or without the addition of 1.0% methionine. The selenoamino acid content of tissues and the distribution of {sup 75}Se in erythrocyte proteins were determined. In comparison to the rats fed the basal diet without added methionine, significantly more {sup 75}Se-selenocysteine was found in liver and muscle, more {sup 75}Se was found in erythrocyte GSHPx and less {sup 75}Se was found in erythrocyte hemoglobin of rats fed 1.0% methionine. These data suggest that methionine diverts SeMet from incorporation into general proteins and enhances its conversion to selenocysteine for specific selenium-requiring proteins, such as GSHPx.

  20. From yeast to human: exploring the comparative biology of methionine restriction in extending eukaryotic life span.

    PubMed

    McIsaac, R Scott; Lewis, Kaitlyn N; Gibney, Patrick A; Buffenstein, Rochelle

    2016-01-01

    Methionine restriction is a widely reported intervention for increasing life span in several model organisms. Low circulating levels of methionine are evident in the long-lived naked mole-rat, suggesting that it naturally presents with a life-extending phenotype akin to that observed in methionine-restricted animals. Similarly, long-lived dwarf mice also appear to have altered methionine metabolism. The mechanisms underlying methionine-restriction effects on life-span extension, however, remain unknown, as do their potential connections with caloric restriction, another well-established intervention for prolonging life span. Paradoxically, methionine is enriched in proteins expressed in mitochondria and may itself serve an important role in the detoxification of reactive oxygen species and may thereby contribute to delayed aging. Collectively, we highlight the evidence that modulation of the methionine metabolic network can extend life span-from yeast to humans-and explore the evidence that sulfur amino acids and the concomitant transsulfuration pathway play a privileged role in this regard. However, systematic studies in single organisms (particularly those that exhibit extreme longevity) are still required to distinguish the fundamental principles concerning the role of methionine and other amino acids in regulating life span. PMID:26995762

  1. From yeast to human: exploring the comparative biology of methionine restriction in extending eukaryotic life span.

    PubMed

    McIsaac, R Scott; Lewis, Kaitlyn N; Gibney, Patrick A; Buffenstein, Rochelle

    2016-01-01

    Methionine restriction is a widely reported intervention for increasing life span in several model organisms. Low circulating levels of methionine are evident in the long-lived naked mole-rat, suggesting that it naturally presents with a life-extending phenotype akin to that observed in methionine-restricted animals. Similarly, long-lived dwarf mice also appear to have altered methionine metabolism. The mechanisms underlying methionine-restriction effects on life-span extension, however, remain unknown, as do their potential connections with caloric restriction, another well-established intervention for prolonging life span. Paradoxically, methionine is enriched in proteins expressed in mitochondria and may itself serve an important role in the detoxification of reactive oxygen species and may thereby contribute to delayed aging. Collectively, we highlight the evidence that modulation of the methionine metabolic network can extend life span-from yeast to humans-and explore the evidence that sulfur amino acids and the concomitant transsulfuration pathway play a privileged role in this regard. However, systematic studies in single organisms (particularly those that exhibit extreme longevity) are still required to distinguish the fundamental principles concerning the role of methionine and other amino acids in regulating life span.

  2. Crystallography captures catalytic steps in human methionine adenosyltransferase enzymes.

    PubMed

    Murray, Ben; Antonyuk, Svetlana V; Marina, Alberto; Lu, Shelly C; Mato, Jose M; Hasnain, S Samar; Rojas, Adriana L

    2016-02-23

    The principal methyl donor of the cell, S-adenosylmethionine (SAMe), is produced by the highly conserved family of methionine adenosyltranferases (MATs) via an ATP-driven process. These enzymes play an important role in the preservation of life, and their dysregulation has been tightly linked to liver and colon cancers. We present crystal structures of human MATα2 containing various bound ligands, providing a "structural movie" of the catalytic steps. High- to atomic-resolution structures reveal the structural elements of the enzyme involved in utilization of the substrates methionine and adenosine and in formation of the product SAMe. MAT enzymes are also able to produce S-adenosylethionine (SAE) from substrate ethionine. Ethionine, an S-ethyl analog of the amino acid methionine, is known to induce steatosis and pancreatitis. We show that SAE occupies the active site in a manner similar to SAMe, confirming that ethionine also uses the same catalytic site to form the product SAE.

  3. Effect of amino acid supplementation on protein quality of soy-based infant formulas fed to rats.

    PubMed

    Sarwar, G; Peace, R W; Botting, H G

    1993-05-01

    The powder forms of soy-based infant formulas obtained from four manufacturers were fed to weanling rats for two weeks, as the sole source of protein in diets containing 8% protein, 20% fat, and adequate amounts of minerals and vitamins. The relative protein efficiency ratio (RPER) and the relative net protein ratio (RNPR) values (casein + methionine = 100) of diets containing unsupplemented formulas were 71-81 and 78-85, respectively. Supplementation of the formula diets with lysine (0.2%), methionine (0.2%), threonine (0.1%) or tryptophan (0.05%) increased the level of the supplemental amino acid in rat serum but generally failed to improve the RPER or RNPR values. Addition of all four essential amino acids to the formula diets, however, caused a marked improvement in their protein quality (RPER or RNPR values = 100). The data suggested that proteins in soy-based formulas could be marginally co-limited in several indispensable amino acids.

  4. Effects of Glycine, Water, Ammonia, and Ammonium Bicarbonate on the Oligomerization of Methionine

    NASA Astrophysics Data System (ADS)

    Huang, Rui; Furukawa, Yoshihiro; Otake, Tsubasa; Kakegawa, Takeshi

    2016-09-01

    The abiotic oligomerization of amino acids may have created primordial, protein-like biological catalysts on the early Earth. Previous studies have proposed and evaluated the potential of diagenesis for the amino acid oligomerization, simulating the formation of peptides that include glycine, alanine, and valine, separately. However, whether such conditions can promote the formation of peptides composed of multiple amino acids remains unclear. Furthermore, the chemistry of pore water in sediments should affect the oligomerization and degradation of amino acids and oligomers, but these effects have not been studied extensively. In this study, we investigated the effects of water, ammonia, ammonium bicarbonate, pH, and glycine on the oligomerization and degradation of methionine under high pressure (150 MPa) and high temperature conditions (175 °C) for 96 h. Methionine is more difficult to oligomerize than glycine and methionine dimer was formed in the incubation of dry powder of methionine. Methionine oligomers as long as trimers, as well as methionylglycine and glycylmethionine, were formed under every condition with these additional compounds. Among the compounds tested, the oligomerization reaction rate was accelerated by the presence of water and by an increase in pH. Ammonia also increased the oligomerization rate but consumed methionine by side reactions and resulted in the rapid degradation of methionine and its peptides. Similarly, glycine accelerated the oligomerization rate of methionine and the degradation of methionine, producing water, ammonia, and bicarbonate through its decomposition. With Gly, heterogeneous dimers (methionylglycine and glycylmethionine) were formed in greater amounts than with other additional compounds although smaller amount of these heterogeneous dimers were formed with other additional compounds. These results suggest that accelerated reaction rates induced by water and co-existing reactive compounds promote the oligomerization

  5. Regulatory structure of the biosynthetic pathway for the aspartate family of amino acids in Lemna paucicostata Hegelm. 6746, with special reference to the role of aspartokinase

    SciTech Connect

    Giovanelli, J.; Mudd, S.H.; Datko, A.H. )

    1989-08-01

    Comprehensive studies were made with Lemna paucicostate Hegelm. 6746 of the effects of combinations of lysine, methionine, and threonine on growth rates, soluble amino acid contents, aspartokinase activities, and fluxes of 4-carbon moieties from aspartate through the aspartokinase step into the amino acids of the aspartate family. These studies show that flux in vitro through the aspartokinase step is insensitive to inhibition by lysine or threonine, and confirm previous in vitro data in establishing that aspartokinase in vivo is present in two orders of magnitude excess of its requirements. No evidence of channeling of the products of the lysine- and threonine-sensitive aspartokinases was obtained, either form of the enzyme along being more than adequate for the combined in vivo flux through the aspartokinase step. The marked insensitivity of flux through the aspartokinase step to inhibition by lysine or threonine strongly suggests that inhibition of aspartokinase by these amino acids is not normally a major factor in regulation of entry of 4-carbon units into the aspartate family of amino acids. Direct measurement of fluxes of 4-carbon units demonstrated that: (a) Lysine strongly feedback regulates its own synthesis, probably at the step catalyzed by dihydrodipicolinate synthase. (b) Threonine alone does not regulate its own synthesis in vivo, thereby confirming previous studies of the metabolism of ({sup 14}C)threonine and ({sup 14}C)homoserine in Lemna.

  6. Enhanced anticancer effect of vincristine with methionine infusion after methionine-depleting total parenteral nutrition in tumor-bearing rats.

    PubMed

    Goseki, N; Nagahama, T; Maruyama, M; Endo, M

    1996-02-01

    Methionine-depleting total parenteral nutrition (Met(-) TPN), in which an amino acid solution devoid of L-methionine and L-cysteine is infused, is thought to reduce tumor cell growth through acting as a partial late S-G2 (i.e., late-S and G2 phases) blocker. The antitumor effect of vincristine (VCR), which acts on mitotic phase cells, was examined with methionine infusion immediately after Met(-) TPN in Yoshida sarcoma (YS)-bearing rats. Rats were given Met(-) TPN for 8 days immediately after inoculation with YS cells (days 0 to 8), which was followed by methionine-containing (Met(+)) regular TPN for 3 days (days 9-11) along with intraperitoneal administration of 0.05 mg/kg/day VCR. All rats were then fed solid food and water ad libitum until they died, with 0.1 mg/kg VCR administration on days 12 and 13. As controls, a Met(-) TPN only group, Met(+) TPN groups with and without VCR, and freely fed groups with and without VCR were studied. The progression of YS was markedly suppressed by Met(-) TPN with VCR. The median survival time in days was 25 days, significantly longer (P<0.001) (generalized Wilcoxon's tests) by 11 to 14 days than that of any of the other groups. In conclusion, VCR appears to have greater efficacy as an anticancer agent when administered together with methionine after Met(-) TPN.

  7. High in vivo rates of methionine biosynthesis in transformed human and malignant rat cells auxotrophic for methionine.

    PubMed

    Hoffman, R M; Erbe, R W

    1976-05-01

    Unlike normal cells, malignant rat and two simian virus 40-transformed human cell lines can neither grow nor survive in B12-and folate-supplemented media in which methionine is replaced by homocysteine. Yet three lines of evidence indicate that the malignant and transformed cells synthesize large amounts of methionine endogenously through the reaction catalyzed by 5-methyltetrahydropteroyl-L-glutamate; L-homocysteine S-methyltransferase (EC 2.1.1.13). (1) The activities of this methyltransferase were comparable in extracts of malignant and normal cells. (2) The uptake of radioactive label from [5-14C]methyltetrahydropteroyl-L-glutamic acid (5-Me-H4PteGlu) was at least as great in the malignant cells as in the normals and was nearly totally dependent on the addition of homocysteine, the methyl acceptor; furthermore, 59-84% of the label incorporated by cells was recovered as methionine.

  8. Increasing levels of dietary crystalline methionine affect plasma methionine profiles, ammonia excretion, and the expression of genes related to the hepatic intermediary metabolism in rainbow trout (Oncorhynchus mykiss).

    PubMed

    Rolland, Marine; Skov, Peter V; Larsen, Bodil K; Holm, Jørgen; Gómez-Requeni, Pedro; Dalsgaard, Johanne

    2016-08-01

    Strictly carnivorous fish with high requirements for dietary protein, such as rainbow trout (Oncorhynchus mykiss) are interesting models for studying the role of amino acids as key regulators of intermediary metabolism. Methionine is an essential amino acid for rainbow trout, and works as a signalling factor in different metabolic pathways. The study investigated the effect of increasing dietary methionine intake on the intermediary metabolism in the liver of juvenile rainbow trout. For this purpose, five diets were formulated with increasing methionine levels from 0.60 to 1.29% dry matter. The diets were fed in excess for six weeks before three sampling campaigns carried out successively to elucidate (i) the hepatic expression of selected genes involved in lipid, glucose and amino acid metabolism; (ii) the postprandial ammonia excretion; and (iii) the postprandial plasma methionine concentrations. The transcript levels of enzymes involved in lipid metabolism (fatty acid synthase, glucose 6 phosphate dehydrogenase and carnitine palmitoyl transferase 1 a), gluconeogenesis (fructose-1,6-biphosphatase) and amino acid catabolism (alanine amino transferase and glutamate dehydrogenase) were significantly affected by the increase in dietary methionine. Changes in gene expression reflected to some extent the decrease in ammonia excretion (P=0.022) and in the hepatosomatic index (HSI; P<0.001) when dietary methionine increased. Postprandial plasma methionine concentrations correlated positively with the dietary level (P<0.001) at the different sampling points. The study shows that the expression of several genes related to the hepatic intermediary metabolism in rainbow trout responded in a dose-dependent manner to increasing levels of dietary methionine.

  9. Increasing levels of dietary crystalline methionine affect plasma methionine profiles, ammonia excretion, and the expression of genes related to the hepatic intermediary metabolism in rainbow trout (Oncorhynchus mykiss).

    PubMed

    Rolland, Marine; Skov, Peter V; Larsen, Bodil K; Holm, Jørgen; Gómez-Requeni, Pedro; Dalsgaard, Johanne

    2016-08-01

    Strictly carnivorous fish with high requirements for dietary protein, such as rainbow trout (Oncorhynchus mykiss) are interesting models for studying the role of amino acids as key regulators of intermediary metabolism. Methionine is an essential amino acid for rainbow trout, and works as a signalling factor in different metabolic pathways. The study investigated the effect of increasing dietary methionine intake on the intermediary metabolism in the liver of juvenile rainbow trout. For this purpose, five diets were formulated with increasing methionine levels from 0.60 to 1.29% dry matter. The diets were fed in excess for six weeks before three sampling campaigns carried out successively to elucidate (i) the hepatic expression of selected genes involved in lipid, glucose and amino acid metabolism; (ii) the postprandial ammonia excretion; and (iii) the postprandial plasma methionine concentrations. The transcript levels of enzymes involved in lipid metabolism (fatty acid synthase, glucose 6 phosphate dehydrogenase and carnitine palmitoyl transferase 1 a), gluconeogenesis (fructose-1,6-biphosphatase) and amino acid catabolism (alanine amino transferase and glutamate dehydrogenase) were significantly affected by the increase in dietary methionine. Changes in gene expression reflected to some extent the decrease in ammonia excretion (P=0.022) and in the hepatosomatic index (HSI; P<0.001) when dietary methionine increased. Postprandial plasma methionine concentrations correlated positively with the dietary level (P<0.001) at the different sampling points. The study shows that the expression of several genes related to the hepatic intermediary metabolism in rainbow trout responded in a dose-dependent manner to increasing levels of dietary methionine. PMID:27105833

  10. Synthesis of lysine methyltransferase inhibitors

    PubMed Central

    Hui, Chunngai; Ye, Tao

    2015-01-01

    Lysine methyltransferase which catalyze methylation of histone and non-histone proteins, play a crucial role in diverse biological processes and has emerged as a promising target for the development of various human diseases, including cancer, inflammation, and psychiatric disorders. However, inhibiting lysine methyltransferases selectively has presented many challenges to medicinal chemists. During the past decade, lysine methyltransferase inhibitors covering many different structural classes have been designed and developed. In this review, we describe the development of selective, small-molecule inhibitors of lysine methyltransferases with an emphasis on their discovery and chemical synthesis. We highlight the current state of lysine methyltransferase inhibitors and discuss future directions and opportunities for lysine methyltransferase inhibitor discovery. PMID:26258118

  11. Insights into the regulatory landscape of the lysine riboswitch

    PubMed Central

    Garst, Andrew D.; Porter, Ely B.; Batey, Robert T.

    2012-01-01

    A prevalent means of regulating gene expression in bacteria is by riboswitches found within mRNA leader sequences. Like protein repressors these RNA elements must bind an effector molecule with high specificity against a background of other cellular metabolites of similar chemical structure to elicit the appropriate regulatory response. Current crystal structures of the lysine riboswitch do not provide a complete understanding of selectivity as recognition is substantially mediated through main chain atoms of the amino acid. Using a directed set of lysine analogs and other amino acids, the relative contributions of the polar functional groups to binding affinity and the regulatory response have been determined. Our results reveal that the lysine riboswitch has >1,000-fold specificity for lysine over other amino acids. To achieve this specificity, the aptamer is highly sensitive to the precise placement of the ε-amino group and relatively tolerant of alterations to the main chain functional groups. At low NTP concentrations, we observe good agreement between the half-maximal regulatory activity (T50) and the affinity of the receptor for lysine (KD) as well many of its analogs. However, above 400 µM [NTP] the concentration of lysine required to elicit transcription termination rises, moving into the riboswitch into a kinetic control regime. These data demonstrate that under physiologically relevant conditions riboswitches can integrate both effector and NTP concentrations to generate a regulatory response appropriate for global metabolic state of the cell. PMID:22771573

  12. Insights into the regulatory landscape of the lysine riboswitch.

    PubMed

    Garst, Andrew D; Porter, Ely B; Batey, Robert T

    2012-10-12

    A prevalent means of regulating gene expression in bacteria is by riboswitches found within mRNA leader sequences. Like protein repressors, these RNA elements must bind an effector molecule with high specificity against a background of other cellular metabolites of similar chemical structure to elicit the appropriate regulatory response. Current crystal structures of the lysine riboswitch do not provide a complete understanding of selectivity as recognition is substantially mediated through main-chain atoms of the amino acid. Using a directed set of lysine analogs and other amino acids, we have determined the relative contributions of the polar functional groups to binding affinity and the regulatory response. Our results reveal that the lysine riboswitch has >1000-fold specificity for lysine over other amino acids. The aptamer is highly sensitive to the precise placement of the ε-amino group and relatively tolerant of alterations to the main-chain functional groups in order to achieve this specificity. At low nucleotide triphosphate (NTP) concentrations, we observe good agreement between the half-maximal regulatory activity (T(50)) and the affinity of the receptor for lysine (K(d)), as well as many of its analogs. However, above 400 μM [NTP], the concentration of lysine required to elicit transcription termination rises, moving into the riboswitch into a kinetic control regime. These data demonstrate that, under physiologically relevant conditions, riboswitches can integrate both effector and NTP concentrations to generate a regulatory response appropriate for global metabolic state of the cell. PMID:22771573

  13. Traumatic Brain Injury Alters Methionine Metabolism: Implications for Pathophysiology

    PubMed Central

    Dash, Pramod K.; Hergenroeder, Georgene W.; Jeter, Cameron B.; Choi, H. Alex; Kobori, Nobuhide; Moore, Anthony N.

    2016-01-01

    Methionine is an essential proteinogenic amino acid that is obtained from the diet. In addition to its requirement for protein biosynthesis, methionine is metabolized to generate metabolites that play key roles in a number of cellular functions. Metabolism of methionine via the transmethylation pathway generates S-adenosylmethionine (SAM) that serves as the principal methyl (−CH3) donor for DNA and histone methyltransferases (MTs) to regulate epigenetic changes in gene expression. SAM is also required for methylation of other cellular proteins that serve various functions and phosphatidylcholine synthesis that participate in cellular signaling. Under conditions of oxidative stress, homocysteine (which is derived from SAM) enters the transsulfuration pathway to generate glutathione, an important cytoprotective molecule against oxidative damage. As both experimental and clinical studies have shown that traumatic brain injury (TBI) alters DNA and histone methylation and causes oxidative stress, we examined if TBI alters the plasma levels of methionine and its metabolites in human patients. Blood samples were collected from healthy volunteers (HV; n = 20) and patients with mild TBI (mTBI; GCS > 12; n = 20) or severe TBI (sTBI; GCS < 8; n = 20) within the first 24 h of injury. The levels of methionine and its metabolites in the plasma samples were analyzed by either liquid chromatography-mass spectrometry or gas chromatography-mass spectrometry (LC-MS or GC-MS). sTBI decreased the levels of methionine, SAM, betaine and 2-methylglycine as compared to HV, indicating a decrease in metabolism through the transmethylation cycle. In addition, precursors for the generation of glutathione, cysteine and glycine were also found to be decreased as were intermediate metabolites of the gamma-glutamyl cycle (gamma-glutamyl amino acids and 5-oxoproline). mTBI also decreased the levels of methionine, α-ketobutyrate, 2 hydroxybutyrate and glycine, albeit to lesser degrees than

  14. The dietary lysine requirement of juvenile hybrid striped bass.

    PubMed

    Griffin, M E; Brown, P B; Grant, A L

    1992-06-01

    Two experiments were conducted to determine the dietary lysine requirement of juvenile hybrid striped bass (Morone saxatilis x M. chrysops). In both experiments the diets contained 35 g crude protein/100 g diet (10 g crude protein supplied by casein and gelatin and 25 g crude protein supplied by crystalline L-amino acids) and contained graded levels of L-lysine.HCl resulting in eight dietary treatments. Diets were fed to triplicate groups of fish and ranged in dietary lysine concentration from 1.2 to 2.6 g/100 g of the dry diet in Experiment 1 and from 0.8 to 2.2 g/100 g of the dry diet in Experiment 2. Weight gain and food efficiency data from Experiment 1 indicated the dietary lysine requirement to be between 1.2 and 1.4 g/100 g of the dry diet. Weight gain, food efficiency and serum lysine data from Experiment 2 confirmed the requirement to be between 1.2 and 1.4 g/100 g of the dry diet. Broken-line analysis of weight gain and food efficiency data from Experiment 2 indicated the dietary lysine requirement to be 1.4 +/- 0.2% of the dry diet, or 4.0 g/100 g of the dietary protein. Changes in the relative proportions of dietary lipid and carbohydrate between the two experiments, although maintaining similar gross energy levels, did not alter the lysine requirement estimate of juvenile hybrid striped bass.

  15. Peptide backbone cleavage by α-amidation is enhanced at methionine residues.

    PubMed

    Hellwig, Michael; Löbmann, Katja; Orywol, Tom

    2015-01-01

    Cleavage reactions at backbone loci are one of the consequences of oxidation of proteins and peptides. During α-amidation, the Cα -N bond in the backbone is cleaved under formation of an N-terminal peptide amide and a C-terminal keto acyl peptide. On the basis of earlier works, a facilitation of α-amidation by the thioether group of adjacent methionine side chains was proposed. This reaction was characterized by using benzoyl methionine and benzoyl alanyl methionine as peptide models. The decomposition of benzoylated amino acids (benzoyl-methionine, benzoyl-alanine, and benzoyl-methionine sulfoxide) to benzamide in the presence of different carbohydrate compounds (reducing sugars, Amadori products, and reductones) was studied during incubation for up to 48 h at 80 °C in acetate-buffered solution (pH 6.0). Small amounts of benzamide (0.3-1.5 mol%) were formed in the presence of all sugars and from all benzoylated species. However, benzamide formation was strongly enhanced, when benzoyl methionine was incubated in the presence of reductones and Amadori compounds (3.5-4.2 mol%). The reaction was found to be intramolecular, because α-amidation of a similar 4-methylbenzoylated amino acid was not enhanced in the presence of benzoyl-methionine and carbohydrate compounds. In the peptide benzoyl-alanyl-methionine, α-amidation at the methionine residue is preferred over α-amidation at the benzoyl peptide bond. We propose here a mechanism for the enhancement of α-amidation at methionine residues.

  16. The Methionine Transamination Pathway Controls Hepatic Glucose Metabolism through Regulation of the GCN5 Acetyltransferase and the PGC-1α Transcriptional Coactivator.

    PubMed

    Tavares, Clint D J; Sharabi, Kfir; Dominy, John E; Lee, Yoonjin; Isasa, Marta; Orozco, Jose M; Jedrychowski, Mark P; Kamenecka, Theodore M; Griffin, Patrick R; Gygi, Steven P; Puigserver, Pere

    2016-05-13

    Methionine is an essential sulfur amino acid that is engaged in key cellular functions such as protein synthesis and is a precursor for critical metabolites involved in maintaining cellular homeostasis. In mammals, in response to nutrient conditions, the liver plays a significant role in regulating methionine concentrations by altering its flux through the transmethylation, transsulfuration, and transamination metabolic pathways. A comprehensive understanding of how hepatic methionine metabolism intersects with other regulatory nutrient signaling and transcriptional events is, however, lacking. Here, we show that methionine and derived-sulfur metabolites in the transamination pathway activate the GCN5 acetyltransferase promoting acetylation of the transcriptional coactivator PGC-1α to control hepatic gluconeogenesis. Methionine was the only essential amino acid that rapidly induced PGC-1α acetylation through activating the GCN5 acetyltransferase. Experiments employing metabolic pathway intermediates revealed that methionine transamination, and not the transmethylation or transsulfuration pathways, contributed to methionine-induced PGC-1α acetylation. Moreover, aminooxyacetic acid, a transaminase inhibitor, was able to potently suppress PGC-1α acetylation stimulated by methionine, which was accompanied by predicted alterations in PGC-1α-mediated gluconeogenic gene expression and glucose production in primary murine hepatocytes. Methionine administration in mice likewise induced hepatic PGC-1α acetylation, suppressed the gluconeogenic gene program, and lowered glycemia, indicating that a similar phenomenon occurs in vivo These results highlight a communication between methionine metabolism and PGC-1α-mediated hepatic gluconeogenesis, suggesting that influencing methionine metabolic flux has the potential to be therapeutically exploited for diabetes treatment.

  17. Maternal obesity disrupts the methionine cycle in baboon pregnancy.

    PubMed

    Nathanielsz, Peter W; Yan, Jian; Green, Ralph; Nijland, Mark; Miller, Joshua W; Wu, Guoyao; McDonald, Thomas J; Caudill, Marie A

    2015-11-01

    Maternal intake of dietary methyl-micronutrients (e.g. folate, choline, betaine and vitamin B-12) during pregnancy is essential for normal maternal and fetal methionine metabolism, and is critical for important metabolic processes including those involved in developmental programming. Maternal obesity and nutrient excess during pregnancy influence developmental programming potentially predisposing adult offspring to a variety of chronic health problems. In the present study, we hypothesized that maternal obesity would dysregulate the maternal and fetal methionine cycle. To test this hypothesis, we developed a nulliparous baboon obesity model fed a high fat, high energy diet (HF-HED) prior to and during gestation, and examined methionine cycle biomarkers (e.g., circulating concentrations of homocysteine, methionine, choline, betaine, key amino acids, folate, and vitamin B-12). Animals were group housed allowing full physical activity and social interaction. Maternal prepregnancy percent body fat was 5% in controls and 19% in HF-HED mothers, while fetal weight was 16% lower in offspring of HF-HED mothers at term. Maternal and fetal homocysteine were higher, while maternal and fetal vitamin B-12 and betaine were lower in the HF-HED group. Elevations in circulating maternal folate were evident in the HF-HED group indicating impaired folate metabolism (methyl-trap) as a consequence of maternal vitamin B-12 depletion. Finally, fetal methionine, glycine, serine, and taurine were lower in the HF-HED fetuses. These data show that maternal obesity disturbs the methionine cycle in primate pregnancy, providing a mechanism for the epigenetic changes observed among obese pregnant women and suggesting diagnostic and therapeutic opportunities in human pregnancies complicated by obesity.

  18. Free amino acid profiling in the giant puffball mushroom (Calvatia gigantea) using UPLC-MS/MS.

    PubMed

    Kıvrak, İbrahim; Kıvrak, Şeyda; Harmandar, Mansur

    2014-09-01

    Wild edible and medicinal mushroom, Calvatia gigantea, was quantitatively analyzed for the determination of its free amino acids using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The concentrations of total free amino acids, essential and non-essential amino acids were 199.65 mg/100 g, 113.69 mg/100 g, and 85.96 mg/100 g in C. gigantea, respectively. This study showed that C. gigantea, so called a giant puffball mushroom, has free amino acids content. The essential amino acids: tryptophan, isoleucine, valine, phenylalanine, leucine, threonine, lysine, histidine, methionine, and the non-essential amino acids: tyrosine, 4-hyrdroxy proline, arginine, proline, glycine, serine, alanine, glutamine, glutamic acid, aspargine, aspartic acid were detected.

  19. Effect of maternal methionine pre-treatment on alcohol-induced exencephaly and axial skeletal dysmorphogenesis in mouse fetuses.

    PubMed

    Padmanabhan, R; Ibrahim, Ahmad; Bener, Abulbari

    2002-02-01

    Alcohol is known to induce folate deficiency and impair methionine synthase activity. Exogenous folic acid (FA) administered periconceptionally has been shown to prevent the first occurrence and recurrence of neural tube defects (NTD) in humans. Since folate, vitamin B(12) and methionine are metabolically interrelated, it was decided to determine the effect of methionine pre-treatment on alcohol-induced NTD and axial skeletal defects in mouse embryos. Following administration of a single dose of 70 or 150 mg/kg of methionine, 0.03 ml/g body weight of ethanol solution (25% v/v of absolute alcohol in saline) was injected intraperitoneally into pregnant mice at critical stages of neural tube development. The controls were either non-treated or saline treated and pair-fed and pair-watered. Fetuses were collected on gestation day 18. Alcohol and methionine plus alcohol numerically enhanced embryonic resorption and induced a significant reduction in fetal body weight. Alcohol alone caused a 3-fold increase in the background frequency of exencephaly in gestation days 7 and 8 treatment groups. The low dose of methionine only numerically reduced the spontaneous exencephaly. Pre-treatment with methionine only produced a numerical but not statistically significant reduction in alcohol-induced exencephaly. The higher dose of methionine did not produce a particularly beneficial effect on embryonic survival, fetal body weight and occurrence of exencephaly. Alcohol-induced cleft palate and limb malformations were ameliorated by methionine pre-treatment. Craniofacial skeleton, vertebrae and ribs were extensively malformed both in the alcohol and methionine plus alcohol groups indicating a lack of rescue effects of methionine. Whereas supernumerary ribs and extra sternal ribs were augmented by methionine, occipitalization of the atlas vertebra was a malformation unique to the pre-treatment group. Plasma levels of several amino acids including that of methionine were significantly

  20. Nutritional consequences of interspecies differences in arginine and lysine metabolism.

    PubMed

    Ball, Ronald O; Urschel, Kristine L; Pencharz, Paul B

    2007-06-01

    Differences in lysine and arginine requirements among various species such as omnivores (humans, pigs, rats, dogs), carnivores (cats), herbivores (rabbits, horses), ruminants (cattle), poultry, and fish, are covered in detail in this article. Although lysine is classified as an indispensable amino acid across species, the classification of arginine as either an indispensable or dispensable amino acid is more ambiguous because of differences among species in rates of de novo arginine synthesis. Because lysine is most often the limiting amino acid in the diet, its requirement has been extensively studied. By use of the ideal protein concept, the requirements of the other indispensable amino acids can be extrapolated from the lysine requirement. The successful use of this concept in pigs is compared with potential application of the ideal protein concept in humans. The current dietary arginine requirement varies widely among species, with ruminants, rabbits, and rats having relatively low requirements and carnivores, fish, and poultry having high requirements. Interspecies differences in metabolic arginine utilization and reasons for different rates of de novo arginine synthesis are reviewed in detail, as these are the primary determinants of the dietary arginine requirement. There is presently no dietary requirement for humans of any age, although this needs to be reassessed, particularly in neonates. A thorough understanding of the factors contributing to the lysine and arginine requirements in different species will be useful in our understanding of human amino acid requirements.

  1. Topological dispositions of lysine. alpha. 380 and lysine. gamma. 486 in the acetylcholine receptor from Torpedo californica

    SciTech Connect

    Dwyer, B.P. )

    1991-04-23

    The locations have been determined, with respect to the plasma membrane, of lysine {alpha}380 and lysine {gamma}486 in the {alpha} subunit and the {gamma} subunit, respectively, of the nicotinic acetylcholine receptor from Torpedo californica. Immunoadsorbents were constructed that recognize the carboxy terminus of the peptide GVKYIAE released by proteolytic digestion from positions 378-384 in the amino acid sequence of the {alpha} subunit of the acetylcholine receptor and the carboxy terminus of the peptide KYVP released by proteolytic digestion from positions 486-489 in the amino acid sequence of the {gamma} subunit. They were used to isolate these peptides from proteolytic digests of polypeptides from the acetylcholine receptor. Sealed vesicles containing the native acetylcholine receptor were labeled with pyridoxal phosphate and sodium ({sup 3}H)-borohydride. The effect of saponin on the incorporation of pyridoxamine phosphate into lysine {alpha}380 and lysine {gamma}486 from the acetylcholine receptor in these vesicles was assessed with the immunoadsorbents. The conclusions that follow from these results are that lysine {alpha}380 is on the inside surface of a vesicle and lysine {gamma}486 is on the outside surface. Because a majority (85%) of the total binding sites for {alpha}-bungarotoxin bind the toxin in the absence of saponin, the majority of the vesicles are right side out with the inside of the vesicle corresponding to the cytoplasmic surface and the outside of the vesicle corresponding to the extracytoplasmic, synaptic surface. Because lysine {alpha}380 and lysine {gamma}486 lie on opposite sides of the membrane, a membrane-spanning segment must be located between the two positions occupied by these two amino acids in the common sequence of a polypeptide of the acetylcholine receptor.

  2. Biofortification of rice with lysine using endogenous histones.

    PubMed

    Wong, H W; Liu, Q; Sun, S S M

    2015-02-01

    Rice is the most consumed cereal grain in the world, but deficient in the essential amino acid lysine. Therefore, people in developing countries with limited food diversity who rely on rice as their major food source may suffer from malnutrition. Biofortification of stable crops by genetic engineering provides a fast and sustainable method to solve this problem. In this study, two endogenous rice lysine-rich histone proteins, RLRH1 and RLRH2, were over-expressed in rice seeds to achieve lysine biofortification. Their protein sequences passed an allergic sequence-based homology test. Their accumulations in rice seeds were raised to a moderate level by the use of a modified rice glutelin 1 promoter with lowered expression strength to avoid the occurrence of physiological abnormalities like unfolded protein response. The expressed proteins were further targeted to protein storage vacuoles for stable storage using a glutelin 1 signal peptide. The lysine content in the transgenic rice seeds was enhanced by up to 35 %, while other essential amino acids remained balanced, meeting the nutritional standards of the World Health Organization. No obvious unfolded protein response was detected. Different degrees of chalkiness, however, were detected in the transgenic seeds, and were positively correlated with both the levels of accumulated protein and lysine enhancement. This study offered a solution to the lysine deficiency in rice, while at the same time addressing concerns about food safety and physiological abnormalities in biofortified crops.

  3. Downregulation of Cdc6 and pre-replication complexes in response to methionine stress in breast cancer cells.

    PubMed

    Booher, Keith; Lin, Da-Wei; Borrego, Stacey L; Kaiser, Peter

    2012-12-01

    Methionine and homocysteine are metabolites in the transmethylation pathway leading to synthesis of the methyl-donor S-adenosylmethionine (SAM). Most cancer cells stop proliferating during methionine stress conditions, when methionine is replaced in the growth media by its immediate metabolic precursor homocysteine (Met-Hcy+). Non-transformed cells proliferate in Met-Hcy+ media, making the methionine metabolic requirement of cancer cells an attractive target for therapy, yet there is relatively little known about the molecular mechanisms governing the methionine stress response in cancer cells. To study this phenomenon in breast cancer cells, we selected methionine-independent-resistant cell lines derived from MDAMB468 breast cancer cells. Resistant cells grew normally in Met-Hcy+ media, whereas their parental MDAMB468 cells rapidly arrest in the G 1 phase. Remarkably, supplementing Met-Hcy+ growth media with S-adenosylmethionine suppressed the cell proliferation defects, indicating that methionine stress is a consequence of SAM limitation rather than low amino acid concentrations. Accordingly, mTORC1 activity, the primary effector responding to amino acid limitation, remained high. However, we found that levels of the replication factor Cdc6 decreased and pre-replication complexes were destabilized in methionine-stressed MDAMB468 but not resistant cells. Our study characterizes metabolite requirements and cell cycle responses that occur during methionine stress in breast cancer cells and helps explain the metabolic uniqueness of cancer cells.

  4. Genetic and biochemical differences in populations bred for extremes in maize grain methionine concentration

    PubMed Central

    2014-01-01

    Background Methionine is an important nutrient in animal feed and several approaches have been developed to increase methionine concentration in maize (Zea mays L.) grain. One approach is through traditional breeding using recurrent selection. Using divergent selection, genetically related populations with extreme differences in grain methionine content were produced. In order to better understand the molecular mechanisms controlling grain methionine content, we examined seed proteins, transcript levels of candidate genes, and genotypes of these populations. Results Two populations were selected for high or low methionine concentration for eight generations and 40 and 56% differences between the high and low populations in grain methionine concentration were observed. Mean values between the high and low methionine populations differed by greater than 1.5 standard deviations in some cycles of selection. Other amino acids and total protein concentration exhibited much smaller changes. In an effort to understand the molecular mechanisms that contribute to these differences, we compared transcript levels of candidate genes encoding high methionine seed storage proteins involved in sulfur assimilation or methionine biosynthesis. In combination, we also explored the genetic mechanisms at the SNP level through implementation of an association analysis. Significant differences in methionine-rich seed storage protein genes were observed in comparisons of high and low methionine populations, while transcripts of seed storage proteins lacking high levels of methionine were unchanged. Seed storage protein levels were consistent with transcript levels. Two genes involved in sulfur assimilation, Cys2 and CgS1 showed substantial differences in allele frequencies when two selected populations were compared to the starting populations. Major genes identified across cycles of selection by a high-stringency association analysis included dzs18, wx, dzs10, and zp27. Conclusions We

  5. Modified bean seed protein phaseolin did not accumulate stably in transgenic tobacco seeds after methionine enhancement mutations

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The major seed storage protein phaseolin of common bean (Phaseolus vulgaris L.) is deficient in methionine, an essential amino acid for human and animal health. To improve the nutritional quality of common bean, we designed methionine enhancement of phaseolin based on the three dimensional structure...

  6. Effects of Graded Levels of Chromium Methionine on Performance, Carcass Traits, Meat Quality, Fatty Acid Profiles of Fat, Tissue Chromium Concentrations, and Antioxidant Status in Growing-Finishing Pigs.

    PubMed

    Tian, Yao-Yao; Gong, Li-Min; Xue, Jian-Xiang; Cao, Jun; Zhang, Li-Ying

    2015-11-01

    A 97-day feeding trial was conducted to investigate the effects of dietary chromium methionine (CrMet) on performance, carcass traits, meat quality, fatty acid profiles of fat, tissue chromium concentrations, and antioxidant status in growing-finishing pigs. A total of 180 crossbred pigs with a mean initial body weight (BW) 30.18 ± 0.28 kg were allotted to 5 treatments with 6 replicates per treatment and 6 pigs per pen in a randomized complete block design based on BW and sex. Treatments were added with 0 (control), 100, 200, 400, and 800 μg/kg chromium as CrMet. Blood samples were obtained from the anterior vena cava on days 97. Carcass characteristics, pork quality, and tissue chromium concentration data were collected from one pig per pen. The results indicated that supplemental CrMet did not significantly affect growth performance, carcass traits, or meat amino acid profiles. Chromium at 100, 400, and 800 μg/kg decreased drip loss but increased shear force (P < 0.05). Pigs fed 100 or 400 μg/kg had a higher 24-h pH than the control (P < 0.05). While meat color, muscle moisture, crude protein, or crude fat were not affected by CrMet. Supplemental 800 μg/kg chromium reduced C18:0 levels in belly fat (P < 0.05), and chromium supplementation increased cis-9, trans 11-conjugated linoleic acid levels linearly (P < 0.05). Dietary CrMet supplementation increased serum, kidney, and muscle chromium contents (P < 0.05) but did not affect liver chromium contents. Besides, tissue chromium concentrations were increased linearly with increased chromium dosage (P < 0.05). Chromium at 400 μg/kg increased serum glutathione peroxidase activities (P < 0.05), and chromium at 800 μg/kg decreased serum total antioxidant capacity levels (P < 0.05). Nevertheless, liver and kidney antioxidant status were not significantly affected by CrMet. These results indicated that dietary supplementation CrMet did not significantly influence growth

  7. Methionine sulfoximine intensifies cancer anorexia.

    PubMed

    Chance, W T; Zhang, F S; Fischer, J E

    1991-05-01

    Consistent anorexia was first observed 33 days after inoculating Fischer 344 rats with methylcholanthrene-induced sarcoma. Daily treatment of a similar group of rats with the glutamine synthetase inhibitor, methionine sulfoximine, elicited significant reductions of feeding by day 29 at a dose that had no effect on nontumor-bearing rats. Blood concentrations of ammonia were elevated in both groups of tumor-bearing rats and brain ammonia level was increased in the methionine sulfoximine-treated tumor-bearing rats. Forebrain concentrations of tyrosine, tryptophan, DOPAC and 5-HIAA were elevated in both groups of tumor-bearing rats. Since ammonia is detoxified through the glutamine synthetase reaction, these results suggest that blood and brain ammonia concentrations are more important than the neurochemical consequences of ammonia detoxification for the etiology of cancer anorexia.

  8. Technical Note: Methionine, a precursor of methane in living plants

    NASA Astrophysics Data System (ADS)

    Lenhart, K.; Althoff, F.; Greule, M.; Keppler, F.

    2015-03-01

    When terrestrial plants were identified as producers of the greenhouse gas methane, much discussion and debate ensued not only about their contribution to the global methane budget but also with regard to the validity of the observation itself. Although the phenomenon has now become more accepted for both living and dead plants, the mechanism of methane formation in living plants remains to be elucidated and its precursor compounds to be identified. We made use of stable isotope techniques to verify the in vivo formation of methane, and, in order to identify the carbon precursor, 13C positionally labeled organic compounds were employed. Here we show that the amino acid L-methionine acts as a methane precursor in living plants. Employing 13C-labeled methionine clearly identified the sulfur-bound methyl group of methionine as a carbon precursor of methane released from lavender (Lavandula angustifolia). Furthermore, when lavender plants were stressed physically, methane release rates and the stable carbon isotope values of the emitted methane greatly increased. Our results provide additional support that plants possess a mechanism for methane production and suggest that methionine might play an important role in the formation of methane in living plants, particularly under stress conditions.

  9. Technical note: Methionine, a precursor of methane in living plants

    NASA Astrophysics Data System (ADS)

    Lenhart, K.; Althoff, F.; Greule, M.; Keppler, F.

    2014-11-01

    When terrestrial plants were identified as producers of the greenhouse gas methane, much discussion and debate ensued, not only about their contribution to the global methane budget, but also with regard to the validity of the observation itself. Although the phenomenon has now become more accepted for both living and dead plants, the mechanism of methane formation in living plants remains to be elucidated and its precursor compounds identified. We made use of stable isotope techniques to verify in vivo formation of methane and, in order to identify the carbon precursor, 13C-positionally labelled organic compounds were employed. Here we show that the amino acid L-methionine acts as a methane precursor in living plants. Employing 13C-labelled methionine clearly identified the sulphur-bound methyl group of methionine as a carbon precursor of methane released from lavender (Lavandula angustifolia). Furthermore, when lavender plants were stressed physically, methane release rates and the stable carbon isotope values of the emitted methane greatly increased. Our results provide additional support that plants possess a mechanism for methane production and suggest that methionine might play an important role in the formation of methane in living plants, particularly under stress conditions.

  10. (11)C-Methionine uptake in secondary brain epilepsy.

    PubMed

    Lopci, E; Bello, L; Chiti, A

    2014-01-01

    Carbon-11 methionine ((11)C-Methionine) is a radio-labeled amino acid currently utilized in Positron Emission Tomography (PET) for imaging primary and metastatic brain tumors. Its clinical use relies mostly on oncologic applications, but the tracer has the potential to investigate other non-malignant conditions. So far, very limited evidence concerns the use of (11)C-Methionine in patients suffering from seizure; however, the tracer can find a proper utilization in this setting especially as a diagnostic complement to (18)F-Fluorodeoxyglucose ((18)F-FDG). Herein we report the case of a 57-year-old patient presenting with epileptic crises secondary to a brain metastasis from bladder carcinoma, who was investigated in our institution with (11)C-Methionine PET. The scan documented the disease recurrence in the left parietal lobe associated with a diffused tracer uptake in the surrounding cerebral circumvolutions, derived from the comitial status. After surgical removal of the metastatic lesion, the patient experienced a complete recovery of symptoms and no further onset of secondary seizure.

  11. Effects of amino acids on the physiochemical properties of potato starch.

    PubMed

    Cui, Min; Fang, Ling; Zhou, Hongxian; Yang, Hong

    2014-05-15

    The objective of this study was to evaluate effects of different amino acid additives (phenylalanine (Phe), methionine (Met), lysine (Lys), arginine (Arg), aspartic acid (Asp) and glutamic acid (Glu)) on the physicochemical properties of potato starch gels. Charge-carrying amino acids (Lys, Arg, Asp and Glu) significantly decreased the swelling power, solubility, light transmittance, L(∗) value and gel strength of potato starch, but increased syneresis during freeze-thaw treatment, while neutral amino acids (Phe and Met) did not cause modifications in starch gels. During heating, potato starch with fortified charge-carrying amino acids showed a lower peak G' (storage modulus), when compared with Phe and Met. Results showed that charge-carrying amino acids could modify physicochemical properties and improve the nutritional values of starch-based products.

  12. Biodegradable tri-block copolymer poly(lactic acid)-poly(ethylene glycol)-poly(l-lysine)(PLA-PEG-PLL) as a non-viral vector to enhance gene transfection.

    PubMed

    Fu, Chunhua; Sun, Xiaoli; Liu, Donghua; Chen, Zhijing; Lu, Zaijun; Zhang, Na

    2011-02-23

    Low cytotoxicity and high gene transfection efficiency are critical issues in designing current non-viral gene delivery vectors. The purpose of the present work was to synthesize the novel biodegradable poly (lactic acid)-poly(ethylene glycol)-poly(l-lysine) (PLA-PEG-PLL) copolymer, and explore its applicability and feasibility as a non-viral vector for gene transport. PLA-PEG-PLL was obtained by the ring-opening polymerization of Lys(Z)-NCA onto amine-terminated NH(2)-PEG-PLA, then acidolysis to remove benzyloxycarbonyl. The tri-block copolymer PLA-PEG-PLL combined the characters of cationic polymer PLL, PLA and PEG: the self-assembled nanoparticles (NPs) possessed a PEG loop structure to increase the stability, hydrophobic PLA segments as the core, and the primary ɛ-amine groups of lysine in PLL to electrostatically interact with negatively charged phosphate groups of DNA to deposit with the PLA core. The physicochemical properties (morphology, particle size and surface charge) and the biological properties (protection from nuclease degradation, plasma stability, in vitro cytotoxicity, and in vitro transfection ability in HeLa and HepG2 cells) of the gene-loaded PLA-PEG-PLL nanoparticles (PLA-PEG-PLL NPs) were evaluated, respectively. Agarose gel electrophoresis assay confirmed that the PLA-PEG-PLL NPs could condense DNA thoroughly and protect DNA from nuclease degradation. Initial experiments showed that PLA-PEG-PLL NPs/DNA complexes exhibited almost no toxicity and higher gene expression (up to 21.64% in HepG2 cells and 31.63% in HeLa cells) than PEI/DNA complexes (14.01% and 24.22%). These results revealed that the biodegradable tri-block copolymer PLA-PEG-PLL might be a very attractive candidate as a non-viral vector and might alleviate the drawbacks of the conventional cationic vectors/DNA complexes for gene delivery in vivo.

  13. Global Profiling of Protein Lysine Malonylation in Escherichia coli Reveals Its Role in Energy Metabolism.

    PubMed

    Qian, Lili; Nie, Litong; Chen, Ming; Liu, Ping; Zhu, Jun; Zhai, Linhui; Tao, Sheng-Ce; Cheng, Zhongyi; Zhao, Yingming; Tan, Minjia

    2016-06-01

    Protein lysine malonylation is a recently identified post-translational modification (PTM), which is evolutionarily conserved from bacteria to mammals. Although analysis of lysine malonylome in mammalians suggested that this modification was related to energy metabolism, the substrates and biological roles of malonylation in prokaryotes are still poorly understood. In this study, we performed qualitative and quantitative analyses to globally identify lysine malonylation substrates in Escherichia coli. We identified 1745 malonylation sites in 594 proteins in E. coli, representing the first and largest malonylome data set in prokaryotes up to date. Bioinformatic analyses showed that lysine malonylation was significantly enriched in protein translation, energy metabolism pathways and fatty acid biosynthesis, implying the potential roles of protein malonylation in bacterial physiology. Quantitative proteomics by fatty acid synthase inhibition in both auxotrophic and prototrophic E. coli strains revealed that lysine malonylation is closely associated with E. coli fatty acid metabolism. Protein structural analysis and mutagenesis experiment suggested malonylation could impact enzymatic activity of citrate synthase, a key enzyme in citric acid (TCA) cycle. Further comparative analysis among lysine malonylome, succinylome and acetylome data showed that these three modifications could participate in some similar enriched metabolism pathways, but they could also possibly play distinct roles such as in fatty acid synthesis. These data expanded our knowledge of lysine malonylation in prokaryotes, providing a resource for functional study of lysine malonylation in bacteria. PMID:27183143

  14. Degradation signals in the lysine-asparagine sequence space.

    PubMed

    Suzuki, T; Varshavsky, A

    1999-11-01

    The N-degrons, a set of degradation signals recognized by the N-end rule pathway, comprise a protein's destabilizing N-terminal residue and an internal lysine residue. We show that the strength of an N-degron can be markedly increased, without loss of specificity, through the addition of lysine residues. A nearly exhaustive screen was carried out for N-degrons in the lysine (K)-asparagine (N) sequence space of the 14-residue peptides containing either K or N (16 384 different sequences). Of these sequences, 68 were found to function as N-degrons, and three of them were at least as active and specific as any of the previously known N-degrons. All 68 K/N-based N-degrons lacked the lysine at position 2, and all three of the strongest N-degrons contained lysines at positions 3 and 15. The results support a model of the targeting mechanism in which the binding of the E3-E2 complex to the substrate's destabilizing N-terminal residue is followed by a stochastic search for a sterically suitable lysine residue. Our strategy of screening a small library that encompasses the entire sequence space of two amino acids should be of use in many settings, including studies of protein targeting and folding. PMID:10545113

  15. Characterization and expression profile of complete functional domain of granulysin/NK-lysin homologue (buffalo-lysin) gene of water buffalo (Bubalus bubalis).

    PubMed

    Kandasamy, Sukumar; Mitra, Abhijit

    2009-04-15

    Granulysin (GNLY)/NK-lysin (NKL) is an effector antimicrobial cationic peptide expressed in the cytotoxic and natural killer lymphocytes. We report here cDNA sequence (405bp) encoding the complete functional domain of buffalo-lysin (bu-lysin), and its expression profile in the various tissues. The nucleotide sequence of bu-lysin exhibited >85% identity with the bovine lysin. Comparison of the deduced amino acid sequence of bu-lysin with those of GNLY/NKL of different species revealed the conservation of six cysteine (Cys) residues and five alpha helices. Unlike the homologues in other species, bu-lysin composed of 11 positively charged Lys residues as in equine. The expression of bu-lysin mRNA in the in vitro cultured lymphocytes was inducible and increased markedly (p<0.05) in a dose dependant manner when incubated with Concanavalin A (ConA). The expression of bu-lysin mRNA in the different tissues was variable: comparatively higher in the spleen and lymph node, moderate in the uterine endometrium and low in the liver and kidney. These results indicate the existence and active expression of GNLY/NKL homologue in water buffalo having a significant influence in immune response.

  16. Growth hormone signaling is necessary for lifespan extension by dietary methionine.

    PubMed

    Brown-Borg, Holly M; Rakoczy, Sharlene G; Wonderlich, Joseph A; Rojanathammanee, Lalida; Kopchick, John J; Armstrong, Vanessa; Raasakka, Debbie

    2014-12-01

    Growth hormone significantly impacts lifespan in mammals. Mouse longevity is extended when growth hormone (GH) signaling is interrupted but markedly shortened with high-plasma hormone levels. Methionine metabolism is enhanced in growth hormone deficiency, for example, in the Ames dwarf, but suppressed in GH transgenic mice. Methionine intake affects also lifespan, and thus, GH mutant mice and respective wild-type littermates were fed 0.16%, 0.43%, or 1.3% methionine to evaluate the interaction between hormone status and methionine. All wild-type and GH transgenic mice lived longer when fed 0.16% methionine but not when fed higher levels. In contrast, animals without growth hormone signaling due to hormone deficiency or resistance did not respond to altered levels of methionine in terms of lifespan, body weight, or food consumption. Taken together, our results suggest that the presence of growth hormone is necessary to sense dietary methionine changes, thus strongly linking growth and lifespan to amino acid availability.

  17. Compartmentalization and Regulation of Mitochondrial Function by Methionine Sulfoxide Reductases in Yeast

    PubMed Central

    Kaya, Alaattin; Koc, Ahmet; Lee, Byung Cheon; Fomenko, Dmitri E.; Rederstorff, Mathieu; Krol, Alain; Lescure, Alain; Gladyshev, Vadim N.

    2010-01-01

    Elevated levels of reactive oxygen species can damage proteins. Sulfur-containing amino acid residues, cysteine and methionine, are particularly susceptible to such damage. Various enzymes evolved to protect proteins or repair oxidized residues, including methionine sulfoxide reductases MsrA and MsrB, which reduce methionine-S-sulfoxide (Met-SO), and methionine-R-sulfoxide (Met-RO) residues, respectively, back to methionine. Here, we show that MsrA and MsrB are involved in the regulation of mitochondrial function. Saccharomyces cerevisiae mutant cells lacking MsrA, MsrB or both proteins, had normal levels of mitochondria, but lower levels of cytochrome c and fewer respiration-competent mitochondria. The growth of single MsrA or MsrB mutants on respiratory carbon sources was inhibited, and that of the double mutant was severely compromised, indicating impairment of mitochondrial function. Although MsrA and MsrB are thought to have similar roles in oxidative protein repair each targeting a diastereomer of methionine sulfoxide, their deletion resulted in different phenotypes. GFP fusions of MsrA and MsrB showed different localization patterns and primarily localized to cytoplasm and mitochondria, respectively. This finding agreed with compartment-specific enrichment of MsrA and MsrB activities. These results show that oxidative stress contributes to mitochondrial dysfunction through oxidation of methionine residues in proteins located in different cellular compartments. PMID:20799725

  18. Selective Deletion of the Internal Lysine Residue from the Peptide Sequence by Collisional Activation

    NASA Astrophysics Data System (ADS)

    Banerjee, Shibdas; Mazumdar, Shyamalava

    2012-11-01

    The gas-phase peptide ion fragmentation chemistry is always the center of attraction in proteomics to analyze the amino acid sequence of peptides and proteins. In this work, we describe the formation of an anomalous fragment ion, which corresponds to the selective deletion of the internal lysine residue from a series of lysine containing peptides upon collisional activation in the ion trap. We detected several water-loss fragment ions and the maximum number of water molecules lost from a particular fragment ion was equal to the number of lysine residues in that fragment. As a consequence of this water-loss phenomenon, internal lysine residues were found to be deleted from the peptide ion. The N,N-dimethylation of all the amine functional groups of the peptide stopped the internal lysine deletion reaction, but selective N-terminal α-amino acetylation had no effect on this process indicating involvement of the side chains of the lysine residues. The detailed mechanism of the lysine deletion was investigated by multistage CID of the modified and unmodified peptides, by isotope labeling and by energy resolved CID studies. The results suggest that the lysine deletion might occur through a unimolecular multistep mechanism involving a seven-membered cyclic imine intermediate formed by the loss of water from a lysine residue in the protonated peptide. This intermediate subsequently undergoes degradation reaction to deplete the interior imine ring from the peptide backbone leading to the deletion of an internal lysine residue.

  19. Metabolism of methionine in the newborn infant: response to the parenteral and enteral administration of nutrients.

    PubMed

    Thomas, Biju; Gruca, Lourdes L; Bennett, Carole; Parimi, Prabhu S; Hanson, Richard W; Kalhan, Satish C

    2008-10-01

    The rates of transmethylation and transsulfuration of methionine were quantified using [1-(13)C]methionine and [C2H3]methionine tracers in newborn infants born at term gestation and in prematurely born low birth weight infants. Whole body rate of protein breakdown was also measured using [2H5]phenylalanine. The response to enteral formula feeding and parenteral nutrition was examined in full term and prematurely born babies, respectively. The relative rates of appearance of methionine and phenylalanine were comparable to the amino acid composition of mixed body proteins. Rates of transmethylation were high, both in full term infants (fast 32 +/- 14 micromol kg(-1) x h(-1); fed 21.7 +/- 3.2) and in preterm infants (57.2 +/- 14.8). Significant flux through the transsulfuration pathway was evident (full term: fast 6.0 +/- 4.4, fed 4.1 +/- 2.1; preterm: 24.9 +/- 9.9 micromol kg(-1) x h(-1)). Transsulfuration of methionine is evident in the human newborn in the immediate neonatal period, suggesting that cysteine may not be considered a "conditionally" essential amino acid for the neonate. The high rate of transmethylation may reflect the high methylation demand, whereas high rates of transsulfuration in premature babies may be related to high demands for glutathione and to the amounts of methionine in parenteral amino acid mixtures.

  20. Basis for the equilibrium constant in the interconversion of l-lysine and l-beta-lysine by lysine 2,3-aminomutase.

    PubMed

    Chen, Dawei; Tanem, Justinn; Frey, Perry A

    2007-02-01

    l-beta-lysine and beta-glutamate are produced by the actions of lysine 2,3-aminomutase and glutamate 2,3-aminomutase, respectively. The pK(a) values have been titrimetrically measured and are for l-beta-lysine: pK(1)=3.25 (carboxyl), pK(2)=9.30 (beta-aminium), and pK(3)=10.5 (epsilon-aminium). For beta-glutamate the values are pK(1)=3.13 (carboxyl), pK(2)=3.73 (carboxyl), and pK(3)=10.1 (beta-aminium). The equilibrium constants for reactions of 2,3-aminomutases favor the beta-isomers. The pH and temperature dependencies of K(eq) have been measured for the reaction of lysine 2,3-aminomutase to determine the basis for preferential formation of beta-lysine. The value of K(eq) (8.5 at 37 degrees C) is independent of pH between pH 6 and pH 11; ruling out differences in pK-values as the basis for the equilibrium constant. The K(eq)-value is temperature-dependent and ranges from 10.9 at 4 degrees C to 6.8 at 65 degrees C. The linear van't Hoff plot shows the reaction to be enthalpy-driven, with DeltaH degrees =-1.4 kcal mol(-1) and DeltaS degrees =-0.25 cal deg(-1) mol(-1). Exothermicity is attributed to the greater strength of the bond C(beta)-N(beta) in l-beta-lysine than C(alpha)-N(alpha) in l-lysine, and this should hold for other amino acids.

  1. Methionine uptake in Corynebacterium glutamicum by MetQNI and by MetPS, a novel methionine and alanine importer of the NSS neurotransmitter transporter family.

    PubMed

    Trötschel, Christian; Follmann, Martin; Nettekoven, Jeannine A; Mohrbach, Tobias; Forrest, Lucy R; Burkovski, Andreas; Marin, Kay; Krämer, Reinhard

    2008-12-01

    The soil bacterium Corynebacterium glutamicum is a model organism in amino acid biotechnology. Here we present the identification of two different L-methionine uptake systems including the first characterization of a bacterial secondary methionine carrier. The primary carrier MetQNI is a high affinity ABC-type transporter specific for l-methionine. Its expression is under the control of the transcription factor McbR, the global regulator of sulfur metabolism in C. glutamicum. Besides MetQNI, a novel secondary methionine uptake system of the NSS (neurotransmitter:sodium symporter) family was identified and named MetP. The MetP system is characterized by a lower affinity for methionine and uses Na(+) ions for energetic coupling. It is also the main alanine transporter in C. glutamicum and is expressed constitutively. These observations are consistent with models of methionine, alanine, and leucine bound to MetP, derived from the X-ray crystal structure of the LeuT transporter from Aquifex aeolicus. Complementation studies show that MetP consists of two components, a large subunit with 12 predicted transmembrane segments and, surprisingly, an additional subunit with one predicted transmembrane segment only. Thus, this new member of the NSS transporter family adds a novel feature to this class of carriers, namely, the functional dependence on an additional small subunit.

  2. In Salmonella enterica, the Gcn5-related acetyltransferase MddA (formerly YncA) acetylates methionine sulfoximine and methionine sulfone, blocking their toxic effects.

    PubMed

    Hentchel, Kristy L; Escalante-Semerena, Jorge C

    2015-01-01

    Protein and small-molecule acylation reactions are widespread in nature. Many of the enzymes catalyzing acylation reactions belong to the Gcn5-related N-acetyltransferase (GNAT; PF00583) family, named after the yeast Gcn5 protein. The genome of Salmonella enterica serovar Typhimurium LT2 encodes 26 GNATs, 11 of which have no known physiological role. Here, we provide in vivo and in vitro evidence for the role of the MddA (methionine derivative detoxifier; formerly YncA) GNAT in the detoxification of oxidized forms of methionine, including methionine sulfoximine (MSX) and methionine sulfone (MSO). MSX and MSO inhibited the growth of an S. enterica ΔmddA strain unless glutamine or methionine was present in the medium. We used an in vitro spectrophotometric assay and mass spectrometry to show that MddA acetylated MSX and MSO. An mddA(+) strain displayed biphasic growth kinetics in the presence of MSX and glutamine. Deletion of two amino acid transporters (GlnHPQ and MetNIQ) in a ΔmddA strain restored growth in the presence of MSX. Notably, MSO was transported by GlnHPQ but not by MetNIQ. In summary, MddA is the mechanism used by S. enterica to respond to oxidized forms of methionine, which MddA detoxifies by acetyl coenzyme A-dependent acetylation.

  3. In Salmonella enterica, the Gcn5-related acetyltransferase MddA (formerly YncA) acetylates methionine sulfoximine and methionine sulfone, blocking their toxic effects.

    PubMed

    Hentchel, Kristy L; Escalante-Semerena, Jorge C

    2015-01-01

    Protein and small-molecule acylation reactions are widespread in nature. Many of the enzymes catalyzing acylation reactions belong to the Gcn5-related N-acetyltransferase (GNAT; PF00583) family, named after the yeast Gcn5 protein. The genome of Salmonella enterica serovar Typhimurium LT2 encodes 26 GNATs, 11 of which have no known physiological role. Here, we provide in vivo and in vitro evidence for the role of the MddA (methionine derivative detoxifier; formerly YncA) GNAT in the detoxification of oxidized forms of methionine, including methionine sulfoximine (MSX) and methionine sulfone (MSO). MSX and MSO inhibited the growth of an S. enterica ΔmddA strain unless glutamine or methionine was present in the medium. We used an in vitro spectrophotometric assay and mass spectrometry to show that MddA acetylated MSX and MSO. An mddA(+) strain displayed biphasic growth kinetics in the presence of MSX and glutamine. Deletion of two amino acid transporters (GlnHPQ and MetNIQ) in a ΔmddA strain restored growth in the presence of MSX. Notably, MSO was transported by GlnHPQ but not by MetNIQ. In summary, MddA is the mechanism used by S. enterica to respond to oxidized forms of methionine, which MddA detoxifies by acetyl coenzyme A-dependent acetylation. PMID:25368301

  4. Characterization and crystal structure of lysine insensitive Corynebacterium glutamicum dihydrodipicolinate synthase (cDHDPS) protein

    SciTech Connect

    Rice, E.A.; Bannon, G.A.; Glenn, K.C.; Jeong, S.S.; Sturman, E.J.; Rydel, T.J.

    2008-11-21

    The lysine insensitive Corynebacterium glutamicum dihydrodipicolinate synthase enzyme (cDHDPS) was recently successfully introduced into maize plants to enhance the level of lysine in the grain. To better understand lysine insensitivity of the cDHDPS, we expressed, purified, kinetically characterized the protein, and solved its X-ray crystal structure. The cDHDPS enzyme has a fold and overall structure that is highly similar to other DHDPS proteins. A noteworthy feature of the active site is the evidence that the catalytic lysine residue forms a Schiff base adduct with pyruvate. Analyses of the cDHDPS structure in the vicinity of the putative binding site for S-lysine revealed that the allosteric binding site in the Escherichia coli DHDPS protein does not exist in cDHDPS due to three non-conservative amino acids substitutions, and this is likely why cDHDPS is not feedback inhibited by lysine.

  5. Possible evidence of amide bond formation between sinapinic acid and lysine-containing bacterial proteins by matrix-assisted laser desorption/ionization (MALDI) at 355 nm

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We previously reported the apparent formation of matrix adducts of 3,5-dimethoxy-4-hydroxy-cinnamic acid (sinapinic acid or SA) via covalent attachment to disulfide bond-containing proteins (HdeA, HdeB and YbgS) from bacterial cell lysates ionized by matrix-assisted laser desorption/ionization (MALD...

  6. Methionine biosynthesis is essential for infection in the rice blast fungus Magnaporthe oryzae.

    PubMed

    Saint-Macary, Marie Emmanuelle; Barbisan, Crystel; Gagey, Marie Josèphe; Frelin, Océane; Beffa, Roland; Lebrun, Marc Henri; Droux, Michel

    2015-01-01

    Methionine is a sulfur amino acid standing at the crossroads of several biosynthetic pathways. In fungi, the last step of methionine biosynthesis is catalyzed by a cobalamine-independent methionine synthase (Met6, EC 2.1.1.14). In the present work, we studied the role of Met6 in the infection process of the rice blast fungus, Magnaporthe oryzae. To this end MET6 null mutants were obtained by targeted gene replacement. On minimum medium, MET6 null mutants were auxotrophic for methionine. Even when grown in presence of excess methionine, these mutants displayed developmental defects, such as reduced mycelium pigmentation, aerial hypha formation and sporulation. They also displayed characteristic metabolic signatures such as increased levels of cysteine, cystathionine, homocysteine, S-adenosylmethionine, S-adenosylhomocysteine while methionine and glutathione levels remained unchanged. These metabolic perturbations were associated with the over-expression of MgCBS1 involved in the reversed transsulfuration pathway that metabolizes homocysteine into cysteine and MgSAM1 and MgSAHH1 involved in the methyl cycle. This suggests a physiological adaptation of M. oryzae to metabolic defects induced by the loss of Met6, in particular an increase in homocysteine levels. Pathogenicity assays showed that MET6 null mutants were non-pathogenic on both barley and rice leaves. These mutants were defective in appressorium-mediated penetration and invasive infectious growth. These pathogenicity defects were rescued by addition of exogenous methionine and S-methylmethionine. These results show that M. oryzae cannot assimilate sufficient methionine from plant tissues and must synthesize this amino acid de novo to fulfill its sulfur amino acid requirement during infection.

  7. Effect of methyl jasmonate application to grapevine leaves on grape amino acid content.

    PubMed

    Garde-Cerdán, Teresa; Portu, Javier; López, Rosa; Santamaría, Pilar

    2016-07-15

    Over the last few years, considerable attention has been paid to the application of elicitors to vineyard. However, research about the effect of elicitors on grape amino acid content is scarce. Therefore, the aim of this study was to evaluate the influence of foliar application of methyl jasmonate on must amino acid content. Results revealed that total amino acid content was not modified by the application of methyl jasmonate. However, the individual content of certain amino acids was increased as consequence of methyl jasmonate foliar application, i.e., histidine, serine, tryptophan, phenylalanine, tyrosine, asparagine, methionine, and lysine. Among them, phenylalanine content was considerably increased; this amino acid is precursor of phenolic and aromatic compounds. In conclusion, foliar application of methyl jasmonate improved must nitrogen composition. This finding suggests that methyl jasmonate treatment might be conducive to obtain wines of higher quality since must amino acid composition could affect the wine volatile composition and the fermentation kinetics.

  8. Effect of methyl jasmonate application to grapevine leaves on grape amino acid content.

    PubMed

    Garde-Cerdán, Teresa; Portu, Javier; López, Rosa; Santamaría, Pilar

    2016-07-15

    Over the last few years, considerable attention has been paid to the application of elicitors to vineyard. However, research about the effect of elicitors on grape amino acid content is scarce. Therefore, the aim of this study was to evaluate the influence of foliar application of methyl jasmonate on must amino acid content. Results revealed that total amino acid content was not modified by the application of methyl jasmonate. However, the individual content of certain amino acids was increased as consequence of methyl jasmonate foliar application, i.e., histidine, serine, tryptophan, phenylalanine, tyrosine, asparagine, methionine, and lysine. Among them, phenylalanine content was considerably increased; this amino acid is precursor of phenolic and aromatic compounds. In conclusion, foliar application of methyl jasmonate improved must nitrogen composition. This finding suggests that methyl jasmonate treatment might be conducive to obtain wines of higher quality since must amino acid composition could affect the wine volatile composition and the fermentation kinetics. PMID:26948648

  9. Lysine nutrition in swine and the related monogastric animals: muscle protein biosynthesis and beyond.

    PubMed

    Liao, Shengfa F; Wang, Taiji; Regmi, Naresh

    2015-01-01

    Improving feed efficiency of pigs with dietary application of amino acids (AAs) is becoming increasingly important because this practice can not only secure the plasma AA supply for muscle growth but also protect the environment from nitrogen discharge with feces and urine. Lysine, the first limiting AA in typical swine diets, is a substrate for generating body proteins, peptides, and non-peptide molecules, while excess lysine is catabolized as an energy source. From a regulatory standpoint, lysine is at the top level in controlling AA metabolism, and lysine can also affect the metabolism of other nutrients. The effect of lysine on hormone production and activities is reflected by the change of plasma concentrations of insulin and insulin-like growth factor 1. Lysine residues in peptides are important sites for protein post-translational modification involved in epigenetic regulation of gene expression. An inborn error of a cationic AA transporter in humans can lead to a lysinuric protein intolerance condition. Dietary deficiency of lysine will impair animal immunity and elevate animal susceptibility to infectious diseases. Because lysine deficiency has negative impact on animal health and growth performance and it appears that dietary lysine is non-toxic even at a high dose of supplementation, nutritional emphasis should be put on lysine supplementation to avoid its deficiency rather than toxicity. Improvement of muscle growth of monogastric animals such as pigs via dietary lysine supply may be due to a greater increase in protein synthesis rather than a decrease in protein degradation. Nevertheless, the underlying metabolic and molecular mechanisms regarding lysine effect on muscle protein accretion merits further clarification. Future research undertaken to fully elucidate the metabolic and regulatory mechanisms of lysine nutrition could provide a sound scientific foundation necessary for developing novel nutritional strategies to enhance the muscle growth and

  10. Formation of methionine sulfoxide during glycoxidation and lipoxidation of ribonuclease A.

    PubMed

    Brock, Jonathan W C; Ames, Jennifer M; Thorpe, Suzanne R; Baynes, John W

    2007-01-15

    Chemical modification of proteins by reactive oxygen species affects protein structure, function and turnover during aging and chronic disease. Some of this damage is direct, for example by oxidation of amino acids in protein by peroxide or other reactive oxygen species, but autoxidation of ambient carbohydrates and lipids amplifies both the oxidative and chemical damage to protein and leads to formation of advanced glycoxidation and lipoxidation end-products (AGE/ALEs). In previous work, we have observed the oxidation of methionine during glycoxidation and lipoxidation reactions, and in the present work we set out to determine if methionine sulfoxide (MetSO) in protein was a more sensitive indicator of glycoxidative and lipoxidative damage than AGE/ALEs. We also investigated the sites of methionine oxidation in a model protein, ribonuclease A (RNase), in order to determine whether analysis of the site specificity of methionine oxidation in proteins could be used to indicate the source of the oxidative damage, i.e. carbohydrate or lipid. We describe here the development of an LC/MS/MS for quantification of methionine oxidation at specific sites in RNase during glycoxidation or lipoxidation by glucose or arachidonate, respectively. Glycoxidized and lipoxidized RNase were analyzed by tryptic digestion, followed by reversed phase HPLC and mass spectrometric analysis to quantify methionine and methionine sulfoxide containing peptides. We observed that: (1) compared to AGE/ALEs, methionine sulfoxide was a more sensitive biomarker of glycoxidative or lipoxidative damage to proteins; (2) regardless of oxidizable substrate, the relative rate of oxidation of methionine residues in RNase was Met29>Met30>Met13, with Met79 being resistant to oxidation; and (3) arachidonate produced a significantly greater yield of MetSO, compared to glucose. The methods developed here should be useful for assessing a protein's overall exposure to oxidative stress from a variety of sources in

  11. Effect of ruminally protected amino acids on milk yield and composition of Jersey cows fed whole cottonseed.

    PubMed

    Bertrand, J A; Pardue, F E; Jenkins, T C

    1998-08-01

    The objectives of this experiment were to determine whether ruminally protected amino acids (AA) increased milk protein when this content was depressed by the addition of whole cottonseeds in the diets of early lactation Jersey cows. Treatments were 1) a control diet, 2) a diet containing whole cottonseed, and 3) a diet containing whole cottonseed and ruminally protected lysine and methionine. Cows were assigned to treatments at a mean of 7 d postpartum and remained on the experiment for 18 wk. Dry matter intake and yields of milk, milk fat, fat-corrected milk, and energy-corrected milk were not affected by treatment. Milk fat content tended to decrease for cows fed diets containing whole cottonseed. However, the percentages of milk protein, total N, and casein N were depressed by the addition of whole cottonseed and were increased by the addition of ruminally protected AA. Plasma concentrations of methionine, but not lysine, were increased when ruminally protected AA were fed, suggesting that lysine was the most limiting.

  12. Metabolism of excess methionine in the liver of intact rat: an in vivo /sup 2/H NMR study

    SciTech Connect

    London, R.E.; Gabel, S.A.; Funk, A.

    1987-11-03

    L-Methionine is the most toxic amino acid if supplied in excess, and the metabolic basis for this toxicity has been extensively studied, with varying conclusions. It is demonstrated here that in vivo /sup 2/H NMR spectroscopy provides a useful approach to the study of the hepatic metabolism of methionine in the anesthetized rat. Resonances corresponding to administered L-(methyl-/sup 2/H/sub 3/)methionine, and to the transmethylation product sarcosine, are observed during the first 10-min period after an intravenous injection of the labeled methionine, and the time dependence has been followed for a period of 5 h. A third resonance, assigned to the N-trimethyl groups of carnitine, phosphorylcholine, and other metabolites, becomes observable several hours after administration of the deuteriated methionine. In addition, there is a small increase in the intensity of the HDO resonance over the period of the study, which is interpreted to reflect the ultimate oxidation of the labeled sarcosine methyl group via mitochondrial sarcosine dehydrogenase. Additional small /sup 2/H resonances assigned to N/sup 1/-methylhistidine and creatine could be observed in perchloric acid extracts of the livers of rats treated with the deuteriated methionine. Inhibition of the flux through the transmethylation pathway is observed in the rat pretreated with the S-ethyl analogue of methionine, ethionine. These data provide strong support for the importance of glycine transmethylation in the catabolism of excess methionine.

  13. Severe dietary lysine restriction affects growth and body composition and hepatic gene expression for nitrogen metabolism in growing rats.

    PubMed

    Kim, J; Lee, K S; Kwon, D-H; Bong, J J; Jeong, J Y; Nam, Y S; Lee, M S; Liu, X; Baik, M

    2014-02-01

    Dietary lysine restriction may differentially affect body growth and lipid and nitrogen metabolism, depending on the degree of lysine restriction. This study was conducted to examine the effect of dietary lysine restriction on growth and lipid and nitrogen metabolism with two different degree of lysine restriction. Isocaloric amino acid-defined diets containing 1.4% lysine (adequate), 0.70% lysine (50% moderate lysine restriction) and 0.35% lysine (75% severe lysine restriction) were fed from the age of 52 to 77 days for 25 days in male Sprague-Dawley rats. The 75% severe lysine restriction increased (p < 0.05) food intake, but retarded (p < 0.05) growth, increased (p < 0.05) liver and muscle lipid contents and abdominal fat accumulation, increased (p < 0.05) blood urea nitrogen levels and mRNA levels of the serine-synthesizing 3-phosphoglycerate dehydrogenase gene, but decreased (p < 0.05) urea cycle arginase gene mRNA levels. In contrast, the 50% lysine restriction did not significantly (p > 0.05) affect body growth and lipid and nitrogen metabolism. Our results demonstrate that severe 75% lysine restriction has detrimental effects on body growth and deregulate lipid and nitrogen metabolism. PMID:23441935

  14. The relation between L-methionine uptake and sodium in rat small intestine in vitro

    PubMed Central

    Newey, H.; Rampone, A. J.; Smyth, D. H.

    1970-01-01

    1. Uptakes of L-methionine and mannitol by rat jejunum in vitro were measured over test periods from 5 to 120 sec after 30 min pre-test periods in the presence or absence of Na. 2. The initial stage in methionine uptake was dependent on the presence of Na+ and to a lesser extent on the K+ concentration. In contrast mannitol uptake was independent of Na and K. 3. The initial stage in methionine uptake can be reactivated 30-60% within 5 sec by replacing an Na-deficient intestine into an Na-containing medium. 4. Initial methionine uptake was greater with a normal intracellular and low medium Na concentration than with a high medium and low intracellular Na concentration. It is suggested that the intracellular Na concentration is a critical factor, more important than the Na gradient, in determining the rate of amino acid transfer across the luminal membrane. PMID:5501050

  15. Kinetic evidence for separate systems in transport of D- and L-methionine by rat small intestine

    SciTech Connect

    Brachet, P.; Alvarado, F.; Puigserver, A.

    1987-03-01

    The kinetics of D- and L-methionine uptake by rings of everted intestine in vitro are consistent with a saturable Michaelis-Menten component plus a linear, diffusional one. All the data could be fit with a diffusion constant, which was essentially the same, independent of whether it was estimated by iteration or by using the extracellular marker, (/sup 3/H), inulin. Similar results were obtained from in vivo perfusion experiments, except that the diffusional term was negligible. D-(3,4-/sup 14/C)Methionine was found to inhibit L-methionine uptake by intestinal rings according to fully noncompetitive kinetics. Another set of experiments with jejunal brush-border membrane vesicles showed that D-methionine uptake is dependent on a Na/sup +/ gradient and is significantly inhibited by L-(/sup 35/S) methionine and L-prolie, but not by ..beta..-alanine and ..cap alpha..-methylaminoisobutyric acid. The results indicate that, in rat jejunum, D-methionine is taken up through a Na/sup +/-dependent pathway distinct from the neutral amino acid (L-methionine) carrier and from the amino acid (L-proline,..cap alpha..-methylaminoisobutyric acid, ..beta..-alanine) carrier.

  16. Complexation des acides aminés basiques arginine, histidine et lysine avec l'ADN plasmidique en solution aqueuse : participation à la capture de radicaux sous irradiation X à 1,5 keV

    NASA Astrophysics Data System (ADS)

    Tariq Khalil, Talat; Taillefumier, Baptiste; Boulanouar, Omar; Mavon, Christophe; Fromm, Michel

    2016-09-01

    L'environnement chimique de l'ADN en situation biologique est complexe notam-ment en raison de la présence d'histones, protéines nucléaires, associées en quantité approximativement égales à l'ADN pour former la chromatine. Les histones possèdent de nombreux radicaux basiques arginine et lysine chargés positivement et dont la majorité se trouve sur les chaînes émergentes, l'ADN présente quant à lui des charges négatives sur ses groupements phosphates localisés tout au long de la double hélice. Dans cette étude, la complexité de la structure de la chromatine nucléaire est dans un premier temps mimée en solution aqueuse par la formation de complexes entre un ADN plasmidique sonde et les trois acides aminés basiques, Arg, His, Lys, qui, mis à part His, sont protonés au pH physiologique. Ces acides aminés libres en solution sont réputés être des capteurs efficaces de radicaux libres, notamment pour le radical hydroxyle, conférant ainsi un pouvoir protecteur vis-à-vis des effets indirects sur l'ADN en situation d'exposition aux rayonnements ionisants. A concentration fixée, les capacités de capture des acides aminés libres, σ, pour le radical hydroxyle sont typiquement les suivantes σHis ≈σArg > σLys (σLys ≈ 0,1 × σArg). Nous avons mesuré les taux de cassures simple brin par plasmide et par Gray (χ) lors d'expositions de solutions aqueuses de complexes [acide aminé - ADN plasmidique] aux rayons X ultra-mous (1,5 keV). A concentrations égales, les trois acides aminés complexés et présents en large excès ne manifestent pas une capacité de protection de l'ADN proportionnelle à leur capacité de capture libre et en solution ; on trouve en effet des taux de cassures dans l'ordre suivant χHis > χArg > χLys (χLys ≈ 0,01 χArg). Après avoir détaillé le mode opératoire de ces mesures, nous analyserons sur des bases bibliographiques, les modes spécifiques d'interaction des acides aminés basiques avec l'ADN. La sp

  17. Changes in amino acids and lipids during embryogenesis of European lobster, Homarus gammarus (Crustacea: Decapoda).

    PubMed

    Rosa, R; Calado, R; Andrade, A M; Narciso, L; Nunes, M L

    2005-02-01

    We studied the amino acid and lipid dynamics during embryogenesis of Homarus gammarus. Major essential amino acids (EAA) in the last stage of embryonic development were arginine, lysine and leucine; major nonessential amino acids (NEAA) were glutamic acid, aspartic acid, valine and glycine. The highest percent of utilization occurred in respect to EAA (27.8%), mainly due to a significant decrease (p<0.05) of methionine (38.3%) and threonine (36.0%). NEAA also decreased significantly (p<0.05, 11.4%), namely serine (38.1%), tyrosine (26.4%) and glutamic acid (25.7%). In contrast, the free amino acid content increased significantly (p<0.05) during embryonic development, especially the free nonessential amino acids (FNEAA). In the last stage, the most abundant FNEAA were glycine, proline, alanine and taurine, and the major free essential amino acids (FEAA) were arginine, lysine and leucine. Lipid content decreased significantly (p<0.05) during embryonic development. A substantial decrease in all neutral lipid classes was observed (>80% of utilization). Major fatty acids were 16:0, 18:0, 18:1n-9, 18:2n-6, 18:3n-3, 20:5n-3 and 22:6n-3. Unsaturated (UFA) and saturated fatty acids (SFA) were used up at similar rates (76.5% and 76.3%, respectively). Within UFA, monounsaturates (MUFA) were consumed more than polyunsaturates (PUFA) (82.9% and 67.5%, respectively). PMID:15649771

  18. Methionine production--a critical review.

    PubMed

    Willke, Thomas

    2014-12-01

    This paper presents an updated critical review about several attempts to contribute methionine (Met) to the world market with an emphasis on fermentation processes, especially from natural biological sources. Analytical methods for the determination of methionine are reviewed as well as applications in feed, food, pharmacy, and medicine. Fermentation studies published within the last five decades are elucidated critically, mainly with respect to the sulfur balance, substrate yield, and the analytical validity. From all the published fermentation data, it can be concluded that up to now no more than 5 g/L methionine are achievable without using genetically modified organisms (GMOs). The highest L-methionine concentration from natural sources reached so far amounts to 35 g/L and is published as a patent using a GMO of Escherichia coli. The review closes with a comprehensive overview of the role and activities of global methionine manufacturers. Some current market data is also presented. PMID:25381187

  19. Methionine production--a critical review.

    PubMed

    Willke, Thomas

    2014-12-01

    This paper presents an updated critical review about several attempts to contribute methionine (Met) to the world market with an emphasis on fermentation processes, especially from natural biological sources. Analytical methods for the determination of methionine are reviewed as well as applications in feed, food, pharmacy, and medicine. Fermentation studies published within the last five decades are elucidated critically, mainly with respect to the sulfur balance, substrate yield, and the analytical validity. From all the published fermentation data, it can be concluded that up to now no more than 5 g/L methionine are achievable without using genetically modified organisms (GMOs). The highest L-methionine concentration from natural sources reached so far amounts to 35 g/L and is published as a patent using a GMO of Escherichia coli. The review closes with a comprehensive overview of the role and activities of global methionine manufacturers. Some current market data is also presented.

  20. Effects of dietary supplementation of methionine and its hydroxy analog DL-2-hydroxy-4-methylthiobutanoic acid on growth performance, plasma hormone levels, and the redox status of broiler chickens exposed to high temperatures.

    PubMed

    Willemsen, H; Swennen, Q; Everaert, N; Geraert, P-A; Mercier, Y; Stinckens, A; Decuypere, E; Buyse, J

    2011-10-01

    Heat stress is known to impair performance and to induce oxidative stress in poultry. The aim of the present study was to compare the effects of dietary supplementation of dl-methionine (dl-M) or the synthetic analog 2-hydroxy-4-methylthiobutanoic acid (dl-HMTBA) on broiler growth performance, plasma hormone levels, and some oxidative stress-related parameters under conditions of chronic exposure to high temperatures (HT). From 2 to 6 wk of age, male broiler chickens were reared under either a constant temperature of 32°C until 6 wk of age or a normal temperature scheme (gradual decrease to 18°C at 5 wk of age). Chicks in both the normal and HT treatments were provided with a commercial grower diet supplemented with either 1.0 or 1.2 g/kg of dl-M or 1.0 or 1.2 g/kg of dl-HMTBA. Because there were no effects of supplement dose, data were pooled over both doses within each temperature treatment. The chronic HT treatment impaired feed intake and BW gain, but these negative effects were less pronounced when the chickens received dl-HMTBA. Exposure to HT was also associated with decreased (P < 0.001) plasma thyroid hormones and increased (P < 0.0001) plasma corticosterone levels. At 4 wk of age, and irrespective of the supplemental source, chickens subjected to HT were characterized by significantly lower plasma TBA-reactive substance levels. In contrast, at 6 wk of age, plasma TBA-reactive substance levels were significantly increased by HT, but this effect was observed only for the chickens receiving dl-M and not for those receiving dl-HMTBA. High temperatures induced a significant increase in hepatic total glutathione (GSH) and oxidized GSH levels, regardless of the supplemental source. However, the hepatic ratios of reduced GSH to total GSH and reduced GSH to oxidized GSH were highest in chickens supplemented with dl-HMTBA. In conclusion, dl-HMTBA supplementation partially prevented the growth-depressing effects of chronic heat exposure compared with dl

  1. Estimation of the standardized ileal digestible valine to lysine ratio required for 25- to 120-kilogram pigs fed low crude protein diets supplemented with crystalline amino acids.

    PubMed

    Liu, X T; Ma, W F; Zeng, X F; Xie, C Y; Thacker, P A; Htoo, J K; Qiao, S Y

    2015-10-01

    Four 28-d experiments were conducted to determine the standardized ileal digestible (SID) valine (Val) to lysine (Lys) ratio required for 26- to 46- (Exp. 1), 49- to 70- (Exp. 2), 71- to 92- (Exp. 3), and 94- to 119-kg (Exp. 4) pigs fed low CP diets supplemented with crystalline AA. The first 3 experiments utilized 150 pigs (Duroc × Landrace × Large White), while Exp. 4 utilized 90 finishing pigs. Pigs in all 4 experiments were randomly allocated to 1 of 5 diets with 6 pens per treatment (3 pens of barrows and 3 pens of gilts) and 5 pigs per pen for the first 3 experiments and 3 pigs per pen for Exp. 4. Diets for all experiments were formulated to contain SID Val to Lys ratios of 0.55, 0.60, 0.65, 0.70, or 0.75. In Exp. 1 (26 to 46 kg), ADG increased (linear, = 0.039; quadratic, = 0.042) with an increasing dietary Val:Lys ratio. The SID Val:Lys ratio to maximize ADG was 0.62 using a linear broken-line model and 0.71 using a quadratic model. In Exp. 2 (49 to 70 kg), ADG increased (linear, = 0.021; quadratic, = 0.042) as the SID Val:Lys ratio increased. G:F improved (linear, = 0.039) and serum urea nitrogen (SUN) decreased (linear, = 0.021; quadratic, = 0.024) with an increased SID Val:Lys ratio. The SID Val:Lys ratios to maximize ADG as well as to minimize SUN levels were 0.67 and 0.65, respectively, using a linear broken-line model and 0.72 and 0.71, respectively, using a quadratic model. In Exp. 3 (71 to 92 kg), ADG increased (linear, = 0.007; quadratic, = 0.022) and SUN decreased (linear, = 0.011; quadratic, = 0.034) as the dietary SID Val:Lys ratio increased. The SID Val:Lys ratios to maximize ADG as well as to minimize SUN levels were 0.67 and 0.67, respectively, using a linear broken-line model and 0.72 and 0.74, respectively, using a quadratic model. In Exp. 4 (94 to 119 kg), ADG increased (linear, = 0.041) and G:F was improved (linear, = 0.004; quadratic, = 0.005) as the dietary SID Val:Lys ratio increased. The SID Val:Lys ratio to maximize G:F was 0

  2. Poly(l-lysine)-graft-folic acid-coupled poly(2-methyl-2-oxazoline) (PLL-g-PMOXA-c-FA): a bioactive copolymer for specific targeting to folate receptor-positive cancer cells.

    PubMed

    Chen, Yin; Cao, Wenbin; Zhou, Junli; Pidhatika, Bidhari; Xiong, Bin; Huang, Lu; Tian, Qian; Shu, Yiwei; Wen, Weijia; Hsing, I-Ming; Wu, Hongkai

    2015-02-01

    In this study, we present the preparation, characterization and application of a novel bioactive copolymer poly(l-lysine)-graft-folic acid-coupled poly(2-methyl-2-oxazoline) (PLL-g-PMOXA-c-FA), which has a specific interaction with folate receptor (FR)-positive cancer cells. Glass surface immobilized with PLL-g-PMOXA-c-FA was demonstrated to be adhesive to FR-positive cancer cells (HeLa, JEG-3) while nonadhesive to FR-negative ones (MCF-7, HepG2) in 3 h. The specific interaction between conjugated FA on the substrate and FRs on the cells could hardly be inhibited unless a high concentration (5 mM) of free FA was used due to the multivalent nature of it. The FA functionality ratio of the copolymer on the substrate had a significant influence on the adhesion of HeLa cells, and our experiments revealed that the affinity of the substrate to the cells declined dramatically with the decrease of functionality ratio. This was believed to be caused by the polydispersity of PMOXA tethers, as supported by GPC and ToF-SIMS data. As a proof of concept in the application of our material, we demonstrated successful recovery of HeLa cells from mixture with MCF-7 (1:100) on the copolymer-coated glass, and our results showed that both high sensitivity (95.6 ± 13.3%) and specificity (24.3 ± 8.6%) were achieved.

  3. Lysine carboxylation in proteins: OXA-10 beta-lactamase.

    PubMed

    Li, Jie; Cross, Jason B; Vreven, Thom; Meroueh, Samy O; Mobashery, Shahriar; Schlegel, H Bernhard

    2005-11-01

    An increasing number of proteins are being shown to have an N(zeta)-carboxylated lysine in their structures, a posttranslational modification of proteins that proceeds without the intervention of a specific enzyme. The role of the carboxylated lysine in these proteins is typically structural (hydrogen bonding or metal coordination). However, carboxylated lysines in the active sites of OXA-10 and OXA-1 beta-lactamases and the sensor domain of BlaR signal-transducer protein serve in proton transfer events required for the functions of these proteins. These examples demonstrate the utility of this unusual amino acid in acid-base chemistry, in expansion of function beyond those of the 20 standard amino acids. In this study, the ONIOM quantum-mechanical/molecular-mechanical (QM/MM) method is used to study the carboxylation of lysine in the OXA-10 beta-lactamase. Lys-70 and the active site of the OXA-10 beta-lactamase were treated with B3LYP/6-31G(d,p) density functional calculations and the remainder of the enzyme with the AMBER molecular mechanics force field. The barriers for unassisted carboxylation of neutral lysine by carbon dioxide or bicarbonate are high. However, when the reaction with CO2 is catalyzed by a molecule of water in the active site, it is exothermic by about 13 kcal/mol, with a barrier of approximately 14 kcal/mol. The calculations show that the carboxylation and decarboxylation of Lys-70 are likely to be accompanied by deprotonation and protonation of the carbamate, respectively. The analysis may also be relevant for other proteins with carboxylated lysines, a feature that may be more common in nature than previously appreciated.

  4. Application of PCDA/SPH/CHO/Lysine vesicles to detect pathogenic bacteria in chicken.

    PubMed

    de Oliveira, Taíla V; Soares, Nilda de F F; de Andrade, Nélio J; Silva, Deusanilde J; Medeiros, Eber Antônio A; Badaró, Amanda T

    2015-04-01

    During the course of infection, Salmonella must successively survive the harsh acid stress of the stomach and multiply into a mild acidic compartment within macrophages. Inducible amino acid decarboxylases are known to promote adaptation to acidic environments, as lysine decarboxylation to cadaverine. The idea of Salmonella defenses responses could be employed in systems as polydiacetylene (PDA) to detect this pathogen so important to public health system. Beside that PDA is an important substance because of the unique optical property; that undergoes a colorimetric transitions by various external stimuli. Therefore 10,12-pentacosadyinoic acid (PCDA)/Sphingomyelin(SPH)/Cholesterol(CHO)/Lysine system was tested to determine the colorimetric response induced by Salmonella choleraesuis. PCDA/SPH/CHO/Lysine vesicles showed a colour change even in low S. choleraesuis concentration present in laboratory conditions and in chicken meat. Thus, this work showed a PCDA/SPH/CHO/Lysine vesicle application to simplify routine analyses in food industry, as chicken meat industry.

  5. [Lysine production by Brevibacterium divaricatum NTU-2 and its recovery from the fermentation broth].

    PubMed

    Su, Y C; Hwang, S M; Huang, J H

    1990-08-01

    An accumulation of L-lysine of about 42 g/l (as L-lysine-HCl) was obtained by cultivating Brevibacterium divaricatum NTU-2 in a medium containing 10.0% glucose, 4.0% (NH4)2 SO4, 0.1% KH2 PO4, 0.04% Mg SO4.7H2 O, 30 ml/l soybean meal hydrolysate, 50 mg/l DL-methionine, 100 micrograms/l d-biotin, 100 micrograms/l thiamine-HCl and 5% CaCO3 at pH 7.0. The yield was about 48.8% based on consumed glucose. The L-lysine accumulated in the broth was recovered and purified by simply using a strong cation-exchange resin, Diaion SK1B. The absorbed L-lysine was eluted with 4% ammonia water, and the average adsorption and desorption rates estimated (by 10 repeated experiments) were found to be 96.1% and 97.5%, respectively. The eluate was concentrated to about 50% by volume and adjusted to pH 4.0 with 8 N HCl. The solution was concentrated again to one half volume and followed by adding three volumes of 95% ethanol. The crude crystals of L-lysine-HCl in purity of 89.5% were obtained by filtration. The recovery was 93.5%. The resulting crude crystals were then dissolved in water, and subjected to decolorization with 0.5% active carbon. The decolorized solution thus obtained was recrystalized by concentration and 95% ethanol addition. After filtration and drying at 80 degrees C for 3 hr, white crystalline L-lysine with purity of 99.2% was finally obtained. The overall recovery of L-lysine was up to 86.7%. PMID:2128693

  6. Biodegradable Tri-Block Copolymer Poly(lactic acid)-poly(ethylene glycol)-poly(l-lysine)(PLA-PEG-PLL) as a Non-Viral Vector to Enhance Gene Transfection

    PubMed Central

    Fu, Chunhua; Sun, Xiaoli; Liu, Donghua; Chen, Zhijing; Lu, Zaijun; Zhang, Na

    2011-01-01

    Low cytotoxicity and high gene transfection efficiency are critical issues in designing current non-viral gene delivery vectors. The purpose of the present work was to synthesize the novel biodegradable poly (lactic acid)-poly(ethylene glycol)-poly(l-lysine) (PLA-PEG-PLL) copolymer, and explore its applicability and feasibility as a non-viral vector for gene transport. PLA-PEG-PLL was obtained by the ring-opening polymerization of Lys(Z)-NCA onto amine-terminated NH2-PEG-PLA, then acidolysis to remove benzyloxycarbonyl. The tri-block copolymer PLA-PEG-PLL combined the characters of cationic polymer PLL, PLA and PEG: the self-assembled nanoparticles (NPs) possessed a PEG loop structure to increase the stability, hydrophobic PLA segments as the core, and the primary ɛ-amine groups of lysine in PLL to electrostatically interact with negatively charged phosphate groups of DNA to deposit with the PLA core. The physicochemical properties (morphology, particle size and surface charge) and the biological properties (protection from nuclease degradation, plasma stability, in vitro cytotoxicity, and in vitro transfection ability in HeLa and HepG2 cells) of the gene-loaded PLA-PEG-PLL nanoparticles (PLA-PEG-PLL NPs) were evaluated, respectively. Agarose gel electrophoresis assay confirmed that the PLA-PEG-PLL NPs could condense DNA thoroughly and protect DNA from nuclease degradation. Initial experiments showed that PLA-PEG-PLL NPs/DNA complexes exhibited almost no toxicity and higher gene expression (up to 21.64% in HepG2 cells and 31.63% in HeLa cells) than PEI/DNA complexes (14.01% and 24.22%). These results revealed that the biodegradable tri-block copolymer PLA-PEG-PLL might be a very attractive candidate as a non-viral vector and might alleviate the drawbacks of the conventional cationic vectors/DNA complexes for gene delivery in vivo. PMID:21541064

  7. Chemical composition and amino acid profiles of goose muscles from native Polish breeds.

    PubMed

    Okruszek, A; Woloszyn, J; Haraf, G; Orkusz, A; Werenska, M

    2013-04-01

    The aim of the study was to compare the chemical and amino acid composition of breast (pectoralis major) and thigh (biceps femoris) muscles in 17-wk-old geese from 2 Polish conservative flocks: Rypińska (Ry, n = 20) and Garbonosa (Ga, n = 20). The geese were fed ad libitum during the experimental period on the same complete feed. Genotypes affected the moisture and fat content of breast and thigh meat. The Ga geese were characterized by higher moisture as well as lower fat lipid content compared with the Ry breast and thigh muscles. The amino acid proportions of meat proteins depended on the goose flock and type of muscles, where significant differences were found. The proteins of Ga breast muscles contained more glutamic acid, glycine, lysine, tryptophan, histidine, and methionine, and less aspartic acid, proline, serine, leucine, valine, phenyloalanine, tyrosine, and threonine than the Ry geese (P ≤ 0.05). The proteins of Ry thigh muscles were characterized by higher content of proline, serine, and essential amino acids (without lysine and methionine) and lower glutamic and asparagine acid, alanine, and glycine compared with the Ga flock. According to the Food and Agriculture Organization of the United Nations/World Health Organization (1991) standard, tryptophan was the amino acid limiting the nutritional value of meat proteins of Ry breast muscles (amino acid score for tryptophan = 90%). Except for tryptophan, the meat proteins of the investigated raw materials contained more essential amino acids than the standard. The total content of essential amino acids for all investigated muscles was also higher (52.51 to 55.54%) than the standard (33.90%). It is evident that muscle protein from both flocks of geese have been characterized by high nutritional value. The values of the essential amino acid index of breast muscle proteins were similar in both flocks.

  8. Lysine fortification: past, present, and future.

    PubMed

    Pellett, Peter L; Ghosh, Shibani

    2004-06-01

    Fortification with lysine to improve the protein value of human diets that are heavily based on cereals has received support from the results of these recent studies [1,2]. Support also comes from examination of average food and nutrient availability data derived from food balance sheets. Whereas nutritional status is influenced by the nutrient content of foods consumed in relation to need, the requirements for protein and amino acids are influenced by many additional factors [10, 12, 14, 28, 29]. These include age, sex, body size, physical activity, growth, pregnancy and lactation, infection, and the efficiency of nutrient utilization. Even if the immune response was influenced by the added lysine, adequate water and basic sanitation would remain essential. Acute and chronic undernutrition and most micronutrient deficiencies primarily affect poor and deprived people who do not have access to food of adequate nutritional value, live in unsanitary environments without access to clean water and basic services, and lack access to appropriate education and information [30]. A further variable is the possible interaction between protein and food energy availability [31]. This could affect the protein value of diets when food energy is limiting to a significant degree. Thus, the additional effects of food energy deficiency on protein utilization could well be superimposed on the very poorest. The improvement of dietary diversity must be the long-term aim, with dietary fortification considered only a short-term solution. The former should take place as wealth improves and the gaps between rich and poor diminish. Although such changes are taking place, they are highly uneven. Over the last several decades, increases have occurred in the availability of food energy, total protein, and animal protein for both developed and developing countries. However, for the very poorest developing countries over the same period, changes have been almost nonexistent, and the values for

  9. Determination of amino acids in fodders and raw materials using capillary zone electrophoresis.

    PubMed

    Komarova, N V; Kamentsev, J S; Solomonova, A P; Anufrieva, R M

    2004-02-01

    Two schemes were offered for analysis of amino acid contents in fodders and raw materials for mixed fodders by capillary zone electrophoresis (CZE). The first variant provides express analysis of four technologically important amino acids (lysine, methionine, threonine, cystine) in borate buffer on characteristic absorption of aminogroup (190 nm), with limits of quantitation being on average 0.2%. The second scheme includes pre-capillary derivatization of amino acids using phenylisothiocyanate (PITC) and separation of phenylthiocarbamyl (PTC)-derivatives obtained by CZE with a detection on 254 nm, which allows to widen a list of detectable components up to 19 (without tryptophan) and significantly improve detection limits down to 0.01%. Acid hydrolysis was used for a sample preparation. The results of analysis of fodders were compared using such methods, as CZE, ion exchange chromatography (amino acid analyzer) and reversed-phase (RP)-HPLC (with gradient technique of elution). PMID:14698247

  10. Effects of methionine supplementation on the incidence of dietary fat induced myocardial lesions in the rat.

    PubMed

    Clandinin, M T; Yamashiro, S

    1980-06-01

    Purified diets were prepared to evaluate the effect of methionine supplementation on the incidence and severity of vegetable oil-induced myocardial lesions in the rat. The unsupplemented basal diet fed was similar in nutrient composition to typical semipurified diets currently utilized for cardiopathogenic evaluation of dietary rapeseed oils and contained 1.276 mg of S-amino acid per kilocalorie. The methionine-supplemented diet contained an additional 0.25% (w/w) L-methionine or a total of 1.815 mg of S-amino acid per kilocalorie. Feeding trials were conducted in which weanling rats were fed either a diet containing 20% (w/w) soybean oil (SBO), low erucic acid rapeseed oil (LER) or high urucic acid rapeseed oil (HER) for 16 or 28 weeks. Dietary supplementation with methionine was found to reduce the incidence of focal myocardial lesions in SBO-fed animals to zero. These results suggest that marginal deficiencies in methionine may interact with the frequency and severity of myocardial changes reported for Sprague-Dawley rats fed various dietary oils. The results indicate that levels of essential nutrients should be adjusted when the energy level of the diet is increased.

  11. Characterization of the fibrinogen binding domain of bacteriophage lysin from Streptococcus mitis.

    PubMed

    Seo, Ho Seong; Sullam, Paul M

    2011-09-01

    The binding of bacteria to human platelets is a likely central mechanism in the pathogenesis of infective endocarditis. Platelet binding by Streptococcus mitis SF100 is mediated in part by a lysin encoded by the lysogenic bacteriophage SM1. In addition to its role in the phage life cycle, lysin mediates the binding of S. mitis to human platelets via its interaction with fibrinogen on the platelet surface. To better define the region of lysin mediating fibrinogen binding, we tested a series of purified lysin truncation variants for their abilities to bind this protein. These studies revealed that the fibrinogen binding domain of lysin is contained within the region spanned by amino acid residues 102 to 198 (lysin(102-198)). This region has no sequence homology to other known fibrinogen binding proteins. Lysin(102-198) bound fibrinogen comparably to full-length lysin and with the same selectivity for the fibrinogen Aα and Bβ chains. Lysin(102-198) also inhibited the binding in vitro of S. mitis to human fibrinogen and platelets. When assessed by platelet aggregometry, the disruption of the lysin gene in SF100 resulted in a significantly longer time to the onset of aggregation of human platelets than that of the parent strain. The preincubation of platelets with purified lysin(102-198) also delayed the onset of aggregation by SF100. These results indicate that the binding of lysin to fibrinogen is mediated by a specific domain of the phage protein and that this interaction is important for both platelet binding and aggregation by S. mitis. PMID:21690235

  12. Recombinant bacteriophage lysins as antibacterials

    PubMed Central

    Fenton, Mark; Ross, Paul; McAuliffe, Olivia; O'Mahony, Jim

    2010-01-01

    With the increasing worldwide prevalence of antibiotic resistant bacteria, bacteriophage endolysins (lysins) represent a very promising novel alternative class of antibacterial in the fight against infectious disease. Lysins are phage-encoded peptidoglycan hydrolases which, when applied exogenously (as purified recombinant proteins) to Gram-positive bacteria, bring about rapid lysis and death of the bacterial cell. A number of studies have recently demonstrated the strong potential of these enzymes in human and veterinary medicine to control and treat pathogens on mucosal surfaces and in systemic infections. They also have potential in diagnostics and detection, bio-defence, elimination of food pathogens and control of phytopathogens. This review discusses the extensive research on recombinant bacteriophage lysins in the context of antibacterials, and looks forward to future development and potential. PMID:21327123

  13. Anti-tumor effect of L-methionine-deprived total parenteral nutrition with 5-fluorouracil administration on Yoshida sarcoma-bearing rats.

    PubMed

    Goseki, N; Endo, M; Onodera, T; Kosaki, G

    1991-07-01

    L-methionine-deprived total parenteral nutrition (methionine-deprived TPN), infusing amino acid solution devoid of L-methionine and L-cysteine by the method of TPN as an only protein source, showed enhancement of the effect of several anti-cancer agents. In this study the combined effect of the methionine-deprived TPN with administration of 5-fluorouracil (5-FU) was examined in Yoshida Sarcoma (YS)-bearing rats, from aspects of effects on the tumor metastasis and the host animal's life span, in the following four groups treated with: methionine-deprived TPN with administration of 5-FU, methionine-deprived TPN without administration of 5-FU, L-methionine-contained TPN plus 5-FU, and L-methionine-contained TPN without 5-FU. In the first experiment, TPN was continued for 8 days in the four groups, and the anti-cancer effect of methionine-deprived TPN and administration of 5-FU based on both the growth of the primary tumor at the implanted site and the tumor metastasis was studied from the view point of pathologic findings of animals killed immediately after these treatments. In experiment 2 the survival period was examined after these treatments for 10 days with subsequent oral feeding until death. The results were as follows: proliferation of YS, transplanted subcutaneously, was markedly suppressed; particularly hematogenous metastasis, characteristic in YS, was prominently blunted then obtained an apparent longer survival period in rats treated with the methionine-deprived TPN with administration of 5-FU.

  14. Intramuscular bioavailability of ketoprofen lysine salt in horses.

    PubMed

    Anfossi, P; Villa, R; Montesissa, C; Carli, S

    1997-06-01

    Lysine salts are often used in human pharmaceuticals to increase the solubility and absorption of acidic drugs when these are administered parenterally. In this study the intramuscular bioavailability of ketoprofen administered as the lysine salt was evaluated in horses (n = 5) treated intravenously and intramuscularly (2.2 mg/kg active substance) in a cross-over study. The absorption rate of ketoprofen administered as the lysine salt was rather low: the mean residence time increased from 31.7 min after IV injection to 128.9 min (after IM injection), and the bioavailability was high (mean 92.4%). The calculated steady state plasma concentrations of ketoprofen during multiple dosage were much higher after intramuscular (0.106 g/ml) than after intravenous (0.066 microgram/ml) administration. Intramuscular injections of the ketoprofen lysine salt can therefore be given to horses, which are particularly prone to develop soft tissue reactions, since use of the lysine salt markedly reduced local irritation at the injection site.

  15. Site-saturation engineering of lysine 47 in cyclodextrin glycosyltransferase from Paenibacillus macerans to enhance substrate specificity towards maltodextrin for enzymatic synthesis of 2-O-D-glucopyranosyl-L-ascorbic acid (AA-2G).

    PubMed

    Han, Ruizhi; Liu, Long; Shin, Hyun-dong; Chen, Rachel R; Du, Guocheng; Chen, Jian

    2013-07-01

    In this work, the site-saturation engineering of lysine 47 in cyclodextrin glycosyltransferase (CGTase) from Paenibacillus macerans was conducted to improve the specificity of CGTase towards maltodextrin, which can be used as a cheap and easily soluble glycosyl donor for the enzymatic synthesis of 2-O-D-glucopyranosyl-L-ascorbic acid (AA-2G) by CGTase. When using maltodextrin as glycosyl donor, four mutants K47F (lysine→ phenylalanine), K47L (lysine→ leucine), K47V (lysine→ valine) and K47W (lysine→ tryptophan) showed higher AA-2G yield as compared with that produced by the wild-type CGTase. The transformation conditions (temperature, pH and the mass ratio of L-ascorbic acid to maltodextrin) were optimized and the highest titer of AA-2G produced by the mutant K47L could reach 1.97 g/l, which was 64.2% higher than that (1.20 g/l) produced by the wild-type CGTase. The reaction kinetics analysis confirmed the enhanced maltodextrin specificity, and it was also found that compared with the wild-type CGTase, the four mutants had relatively lower cyclization activities and higher disproportionation activities, which was favorable for AA-2G synthesis. The mechanism responsible for the enhanced substrate specificity was further explored by structure modeling and it was indicated that the enhancement of maltodextrin specificity may be due to the short residue chain and the removal of hydrogen bonding interactions between the side chain of residue 47 and the sugar at -3 subsite. Here the obtained mutant CGTases, especially the K47L, has a great potential in the production of AA-2G with maltodextrin as a cheap and easily soluble substrate.

  16. Selective Oxidation of Methionine and Tryptophan Residues in a Therapeutic IgG1 Molecule.

    PubMed

    Folzer, Emilien; Diepold, Katharina; Bomans, Katrin; Finkler, Christof; Schmidt, Roland; Bulau, Patrick; Huwyler, Jörg; Mahler, Hanns-Christian; Koulov, Atanas V

    2015-09-01

    Oxidation of methionine and tryptophan are common degradation pathways for monoclonal antibodies and present major analytical challenges in biotechnology. Generally, protein oxidation is detectable in stability and/or stressed samples (e.g., exposed to hydrogen peroxide, UV light, or metal ions). The induced chemical modifications may impact the biological activity of antibodies and may have biological consequences. However, these effects and the contribution of individual protein modifications are difficult to delineate as different amino acids are often oxidized simultaneously and accompanied by other degradants such as aggregates, especially in forced degradation studies. Here, we report a new method to obtain selective oxidation of methionine or tryptophan by using oxidation reagents combined with large excess of free tryptophan or methionine, correspondingly. More specifically, using hydrogen peroxide or tert-butyl hydroperoxide in combination with addition of free tryptophan allowed for selective oxidation of methionine. Conversely, the use of 2,2-azobis(2-amidinopropane) dihydrochloride in combination with free methionine resulted in selective tryptophan oxidation, whereas methionine oxidation was not significantly altered. This novel stress model system may prove to be valuable tool in future mechanistic studies of oxidative degradation of protein therapeutics.

  17. Changes in plasma methionine and total homocysteine levels in patients receiving methotrexate infusions.

    PubMed

    Broxson, E H; Stork, L C; Allen, R H; Stabler, S P; Kolhouse, J F

    1989-11-01

    Methotrexate reduces intracellular pools of 5-methyltetrahydrofolate and could result in reduced conversion of homocysteine to methionine by methionine synthetase. This study was designed to investigate the effects of moderate dose to very high dose methotrexate on methionine and total homocysteine as reflections of methotrexate induced intracellular events. Methionine and total homocysteine were measured prior to, during, and following twenty-six 24-h i.v. infusions of 33.6 g/m2 methotrexate (very high dose methotrexate) in 16 children with acute lymphocytic leukemia and seven 4-h i.v. infusions of 8 g/m2 methotrexate (high dose methotrexate) in 5 children with osteogenic sarcoma. Amino acids were measured by gas chromatography/mass spectrophotometry. Mean methionine levels decreased by 70.0 +/- 3.1% (SE) with very high dose methotrexate and 72.6 +/- 5.9% with high dose methotrexate at 24 and 4.5 h, respectively, after beginning methotrexate infusions. Mean total homocysteine levels increased by 61.7 +/- 3.1% with very high dose methotrexate and 55.6 +/- 17.5% with high dose methotrexate at 36 and 24 h, respectively, after beginning methotrexate infusions. No consistent or significant changes were noted in levels of total cysteine, leucine, isoleucine, or valine. Similar changes did not occur in patients receiving prednisone, vincristine, daunomycin, and intrathecal methotrexate as therapy for acute lymphocytic leukemia. These changes in homocysteine and methionine may reflect biological effects of methotrexate that may predict cytotoxicity of methotrexate.

  18. Methionine metabolism in human pregnancy123

    PubMed Central

    Dasarathy, Jaividhya; Gruca, Lourdes L; Bennett, Carole; Parimi, Prabhu S; Duenas, Clarita; Marczewski, Susan; Fierro, Julie L

    2010-01-01

    Background: Hyperhomocysteinemia during pregnancy, which is a consequence of perturbations in methionine and/or folate metabolism, has been implicated in adverse outcomes such as neural tube defects, preeclampsia, spontaneous abortion, and premature delivery. The adaptive changes in methionine metabolism during pregnancy in humans have not been determined. Objective: Our objective was to examine the kinetics of methionine and its rate of transsulfuration and transmethylation in healthy women with advancing gestation. Design: The whole-body rate of appearance (Ra) of methionine and phenylalanine was measured in healthy pregnant women during the first (n = 10), second (n = 5), and third (n = 10) trimesters of pregnancy. These data were compared with those for nonpregnant women (n = 8). Tracers [1-13C]methionine, [C2H3]methionine, and [2H5]phenylalanine were administered as prime-constant rate infusions. The effect of enteral high-protein, mixed-nutrient load on tracer-determined variables was also examined. Results: In pregnant women, the Ra of phenylalanine was significantly (P < 0.05) lower in the first trimester than in the second and third trimesters and was significantly lower than that in nonpregnant women. A linear positive correlation was evident between gestational age and phenylalanine Ra. The fractional rate and total rate of transsulfuration of methionine was significantly (P < 0.05) higher during the first trimester, whereas the rate of transmethylation was higher during the third trimester. Plasma concentrations of total cysteine and homocysteine were lower during pregnancy. Conclusions: Uncomplicated pregnancy in humans is associated with a higher rate of transsulfuration early in gestation and a higher rate of transmethylation of methionine in late gestation. These data may have implications for understanding the role of methionine and homocysteine in complications of pregnancy and for the nutritional care of pregnant women. PMID:19939983

  19. Mechanism of adenylate kinase. Are the essential lysines essential?

    PubMed

    Tian, G C; Yan, H G; Jiang, R T; Kishi, F; Nakazawa, A; Tsai, M D

    1990-05-01

    Using site-specific mutagenesis, we have probed the structural and functional roles of lysine-21 and lysine-27 of adenylate kinase (AK) from chicken muscle expressed in Escherichia coli. The two residues were chosen since according to the nuclear magnetic resonance (NMR) model [Mildvan, A. S., & Fry, D. C. (1987) Adv. Enzymol. 58, 241-313], they are located near the alpha- and the gamma-phosphates, respectively, of adenosine 5'-triphosphate (ATP) in the AK-MgATP complex. In addition, a lysine residue (Lys-21 in the case of AK) along with a glycine-rich loop is considered "essential" in the catalysis of kinases and other nucleotide binding proteins. The Lys-27 to methionine (K27M) mutant showed only slight increases in kcat and Km, but a substantial increase (1.8 kcal/mol) in the free energy of unfolding, relative to the WT AK. For proper interpretation of the steady-state kinetic data, viscosity-dependent kinetics was used to show that the chemical step is partially rate-limiting in the catalysis of AK. Computer modeling suggested that the folded form of K27M could gain stability (relative to the wild type) via hydrophobic interactions of Met-27 with Val-179 and Phe-183 and/or formation of a charge-transfer complex between Met-27 and Phe-183. The latter was supported by an upfield shift of the methyl protons of Met-27 in 1H NMR. Other than this, the 1H NMR spectrum of K27M is very similar to that of WT, suggesting little perturbation in the global or even local conformations.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2161682

  20. 21 CFR 172.399 - Zinc methionine sulfate.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Zinc methionine sulfate. 172.399 Section 172.399... Additives § 172.399 Zinc methionine sulfate. Zinc methionine sulfate, CAS Reg. No. 56329-42-1, may be safely... reaction between equimolar amounts of zinc sulfate and DL-methionine in purified water. (b) The...

  1. Crystallography captures catalytic steps in human methionine adenosyltransferase enzymes

    PubMed Central

    Murray, Ben; Antonyuk, Svetlana V.; Marina, Alberto; Lu, Shelly C.; Mato, Jose M.; Hasnain, S. Samar; Rojas, Adriana L.

    2016-01-01

    The principal methyl donor of the cell, S-adenosylmethionine (SAMe), is produced by the highly conserved family of methionine adenosyltranferases (MATs) via an ATP-driven process. These enzymes play an important role in the preservation of life, and their dysregulation has been tightly linked to liver and colon cancers. We present crystal structures of human MATα2 containing various bound ligands, providing a “structural movie” of the catalytic steps. High- to atomic-resolution structures reveal the structural elements of the enzyme involved in utilization of the substrates methionine and adenosine and in formation of the product SAMe. MAT enzymes are also able to produce S-adenosylethionine (SAE) from substrate ethionine. Ethionine, an S-ethyl analog of the amino acid methionine, is known to induce steatosis and pancreatitis. We show that SAE occupies the active site in a manner similar to SAMe, confirming that ethionine also uses the same catalytic site to form the product SAE. PMID:26858410

  2. Intermediates in the recycling of 5-methylthioribose to methionine in fruits.

    PubMed

    Kushad, M M; Richardson, D G; Ferro, A J

    1983-10-01

    The recycling of 5-methylthioribose (MTR) to methionine in avocado (Persea americana Mill, cv Hass) and tomato (Lycopersicum esculentum Mill, cv unknown) was examined. [(14)CH(3)]MTR was not metabolized in cell free extract from avocado fruit. Either [(14)CH(3)]MTR plus ATP or [(14)CH(3)]5-methylthioribose-1-phosphate (MTR-1-P) alone, however, were metabolized to two new products by these extracts. MTR kinase activity has previously been detected in these fruit extracts. These data indicate that MTR must be converted to MTR-1-P by MTR kinase before further metabolism can occur. The products of MTR-1-P metabolism were tentatively identified as alpha-keto-gamma-methylthiobutyric acid (alpha-KMB) and alpha-hydroxy-gamma-methylthiobutyric acid (alpha-HMB) by chromatography in several solvent systems. [(35)S]alpha-KMB was found to be further metabolized to methionine and alpha-HMB by these extracts, whereas alpha-HMB was not. However, alpha-HMB inhibited the conversion of alpha-KMB to methionine. Both [U-(14)C]alpha-KMB and [U-(14)C]methionine, but not [U-(14)C]alpha-HMB, were converted to ethylene in tomato pericarp tissue. In addition, aminoethoxyvinylglycine inhibited the conversion of alpha-KMB to ethylene. These data suggest that the recycling pathway leading to ethylene is MTR --> MTR-1-P --> alpha-KMB --> methionine --> S-adenosylmethionine --> 1-aminocyclopropane-1-carboxylic acid --> ethylene.

  3. pH dependent growth of poly( L-lysine)/poly( L-glutamic) acid multilayer films and their cell adhesion properties

    NASA Astrophysics Data System (ADS)

    Richert, Ludovic; Arntz, Youri; Schaaf, Pierre; Voegel, Jean-Claude; Picart, Catherine

    2004-10-01

    The short-term interaction of chondrosarcoma cells with (PGA/PLL) polyelectrolyte multilayers was investigated in a serum-containing medium for films built at different pHs and subsequently exposed to the culture medium. The buildup of the films and their stability was first investigated by means of optical waveguide lightmode spectroscopy, quartz crystal microbalance, streaming potential measurements and atomic force microscopy. While film growth is linear at all pHs, after a few layers have been deposited the growth is much larger for the films built at basic pH and even more pronounced for those built at acidic pH. However, these latter films remain stable in the culture medium only if they have been crosslinked prior to the ionic strength and pH jumps. The films built at acidic pH were found to swell in water by about 200% whereas those built at other pHs did not swell in a physiological buffer. For thin films (≈20 nm) built at pH = 7.4, the detachment forces were dependent on the outermost layer, the forces being significantly higher on PLL-ending films than on PGA-ending ones. In contrast, for the thick films built at pH = 4.4 and at pH = 10.4 (thickness of the order of few hundred of nanometers), the detachment forces were independent of the outermost layer of the film. The films built at pH = 10.4, which shrink in contact with salt containing solutions, were highly cell adhesive whereas those built at acidic pH were highly cell resistant. Protein adsorption and film roughness (as measured by AFM) could not explain these striking differences. The high adhesion observed on the film built at pH 10.4 may rather be related to the secondary structure of the film and to its relatively low swellability in water, whereas the cell resistance of the films built at pH 4.4 may be linked to their high swellability. Therefore, for the PGA/PLL films, the cell adhesion properties can be tuned depending on the deposition pH of the polyelectrolyte solutions. This study

  4. Molecular cloning and characterization of l-methionine γ-lyase from Streptomyces avermitilis.

    PubMed

    Kudou, Daizou; Yasuda, Eri; Hirai, Yoshiyuki; Tamura, Takashi; Inagaki, Kenji

    2015-10-01

    A pyridoxal 5'-phosphate-dependent methionine γ-lyase (MGL) was cloned from Streptomyces avermitilis catalyzed the degradation of methionine to α-ketobutyrate, methanethiol, and ammonia. The sav7062 gene (1,242 bp) was corresponded to 413 amino acid residues with a molecular mass of 42,994 Da. The deduced amino acid sequence showed a high degree of similarity to those of other MGL enzymes. The sav7062 gene was overexpressed in Escherichia coli. The enzyme was purified to homogeneity and exhibited the MGL catalytic activities. We cloned the enzyme that has the MGL activity in Streptomyces for the first time.

  5. Thyroid metabolism in the recessive sex-linked dwarf female chicken. 5. Effects of exogenous thyroid hormones on amino acid uptake by eggs.

    PubMed

    Grandhi, R R; Brown, R G; Reinhart, B S; Summers, J D

    1975-03-01

    The incorporation of L-methionine-3H(G) and L-lysine-4,5'3H(n) into egg albumen by dwarf and non-dwarf White Leghorn hens was studied together with the influence of exogenous triiodothyronine (T3) and tetraiodothyronine (T4) on those incorporation patterns. The results were influenced by the amino acid studied and by the strain of bird. The maximum uptake of methionine was a day 1 for the normal and at day 4 for the dwarf which suggested that the dwarf was storing methionine in the tissue first before incorporation into albumen. If the birds were given T3 the maximum for the normal was at day 1 and day 3 for the dwarfs. When T4 was given the maximum uptake was found at day 5 for normals, and at day 2 for dwarfs. In the dwarf, T4 administration essentially corrected the decreased rate uptake of methionine but markedly retarded its uptake by the normal birds. The results for lysine were quite different. The maximum uptake was on day 4 in control, day 3 for T3-treated and day 1 for T4-treated, normal birds. In dwarf birds, the maximum was at day 2 for the control and T4-treated, and day 4 for T3-treated groups. The data were interpreted to suggest strain differences in amino acid uptake patterns due to different rates of synthesis or composition of egg albumen and marked differences in response or recptor sites to thyroid hormones.

  6. Methionine depletion modulates the antitumor and antimetastatic efficacy of ethionine.

    PubMed

    Guo, H; Tan, Y; Kubota, T; Moossa, A R; Hoffman, R M

    1996-01-01

    The elevated methionine requirement for the growth of tumors, termed methionine dependence, is a potentially highly effective therapeutic target. To attack this target we are developing anti-methionine chemotherapy. In this study of anti-methionine chemotherapy we have observed that the methionine analog ethionine is synergistic with methionine depletion in arresting the growth of the Yoshida sarcoma both in vitro and when transplanted to nude mice. In contrast, ethionine in vitro in a methionine-containing medium is not effective against Yoshida sarcoma cells. Similarly, ethionine administered along with a methionine-containing diet is ineffective against the Yoshida sarcoma growing in nude mice. A methionine-depleted diet alone is only partially effective against tumor growth. The Yoshida sarcoma gave rise to metastases in 75% of the- organs observed in the mice on the methionine-containing diet, and 43 % of the organs in the mice on the methionine-free diet. In striking contrast, no metastases were observed in the ethionine-treated animals on the methionine-free diet. Anti-methionine chemotherapy consisting of dietary methionine depletion and ethionine administration caused an initial weight loss but the animals weight stabilized resulting in no animal deaths. The synergism of ethionine and methionine depletion is markedly similar in vitro and in vivo suggesting the observed efficacy is due to the specific anti-methionine targeting. Thus methionine depletion highly potentiates the anti-tumor and anti-metastatic effectiveness of ethionine suggesting that anti-methionine chemotherapy consisting of methionine depletion as a modulator of methionine analogs holds great promise as a new, tumor-selective therapeutic approach.

  7. A GC-ECD method for estimation of free and bound amino acids, gamma-aminobutyric acid, salicylic acid, and acetyl salicylic acid from Solanum lycopersicum (L.).

    PubMed

    Meher, Hari Charan; Gajbhiye, Vijay T; Singh, Ghanendra

    2011-01-01

    A gas chromatograph with electron capture detection method for estimation of selected metabolites--amino acids (free and bound), gamma-aminobutyric acid (GABA), salicylic acid (SA), and acetyl salicylic acid (ASA) from tomato--is reported. The method is based on nitrophenylation of the metabolites by 1-fluoro-2, 4-dinitrobenzene under aqueous alkaline conditions to form dinitophenyl derivatives. The derivatives were stable under the operating conditions of GC. Analysis of bound amino acids comprised perchloric acid precipitation of protein, alkylation (carboxymethylation) with iodoacetic acid, vapor-phase hydrolysis, and derivatization with 1-fluoro-2,4-dinitrobenzene in that order. The metabolites were resolved in 35 min, using a temperature-programmed run. The method is rapid, sensitive, and precise. It easily measured the typical amino acids (aspartate, asparagine, glutamate, glutamine, alanine, leucine, lysine, and phenylalanine) used for identification and quantification of a protein, resolved amino acids of the same mass (leucine and isoleucine), satisfactorily measured sulfur amino acid (methionine, cystine, and cysteine), and quantified GABA, SA, and ASA, as well. The developed method was validated for specificity, linearity, and precision. It has been applied and recommended for estimation of 25 metabolites from Solanum lycopersicum (L.).

  8. A GC-ECD method for estimation of free and bound amino acids, gamma-aminobutyric acid, salicylic acid, and acetyl salicylic acid from Solanum lycopersicum (L.).

    PubMed

    Meher, Hari Charan; Gajbhiye, Vijay T; Singh, Ghanendra

    2011-01-01

    A gas chromatograph with electron capture detection method for estimation of selected metabolites--amino acids (free and bound), gamma-aminobutyric acid (GABA), salicylic acid (SA), and acetyl salicylic acid (ASA) from tomato--is reported. The method is based on nitrophenylation of the metabolites by 1-fluoro-2, 4-dinitrobenzene under aqueous alkaline conditions to form dinitophenyl derivatives. The derivatives were stable under the operating conditions of GC. Analysis of bound amino acids comprised perchloric acid precipitation of protein, alkylation (carboxymethylation) with iodoacetic acid, vapor-phase hydrolysis, and derivatization with 1-fluoro-2,4-dinitrobenzene in that order. The metabolites were resolved in 35 min, using a temperature-programmed run. The method is rapid, sensitive, and precise. It easily measured the typical amino acids (aspartate, asparagine, glutamate, glutamine, alanine, leucine, lysine, and phenylalanine) used for identification and quantification of a protein, resolved amino acids of the same mass (leucine and isoleucine), satisfactorily measured sulfur amino acid (methionine, cystine, and cysteine), and quantified GABA, SA, and ASA, as well. The developed method was validated for specificity, linearity, and precision. It has been applied and recommended for estimation of 25 metabolites from Solanum lycopersicum (L.). PMID:21391500

  9. Identification of methionine as a possible precursor to the selenocysteine catalytic site of glutathione peroxidase

    SciTech Connect

    Chung, C.K.

    1985-01-01

    The selenium (Se) moiety of glutathione peroxidase (GSHPx) occurs as selenocysteine and is present at the catalytic active site of the enzyme which catalyzes the reduction of hydrogen peroxides and lipid peroxides. The presence of this unusual amino acid at the active site raises the question as to the origin of the carbon skeleton of Se-cysteine. ICR Swiss mice were fed a Se deficient diet for 50 days and then were fed a Se adequate diet (1 ppm Se as SeO/sub 3/). Mice were i.p. injected with either (U-/sup 14/C) methionine, serine, or alanine (0.5 ..mu..Ci/0.1 ml/mouse/day) for 25 days. Recovered GSHPx activity in liver and blood was carboxymethylated (CM) with iodoacetic acid. CM-GSHPx was partially purified by column chromatography. /sup 14/C-GSHPx fractions were collected, lyophilized, and hydrolyzed. /sup 14/C-amino acids were separated by TLC and ion-exchange chromatography. TLC (phenol, cyclohexane, acetic acid, and water (90;6.5;3.5;8)) revealed a GSHPx /sup 14/C-amino acid derived from U-/sup 14/C-methionine, but not from serine or alanine corresponding to CM-selenocysteine (R/sub f/; 0.16). Ion-exchange chromatography of U-/sup 14/C-methionine labeled GSHPx hydrolyzate revealed two radio carbon ninhydrin positive peaks corresponding to /sup 14/C-CM-selenocysteine and /sup 14/C-methionine. No corresponding /sup 14/C-labeled peaks were observed for CM-selenocysteine derived from U-/sup 14/C serine or alanine. The results suggest that methionine may contribute a portion of the carbon skeleton to selenocysteine which may include an alternative metabolic pathway. Animal studies demonstrated that GSHPx activity is increased by methionine supplementation may be due to its contribution of carbon source to the catalytic site of the enzyme.

  10. Light-activated amino acid transport in Halobacterium halobium envelope vesicles

    NASA Technical Reports Server (NTRS)

    Macdonald, R. E.; Lanyi, J. K.

    1977-01-01

    Vesicles prepared from Halobacterium halobium cell envelopes accumulate amino acids in response to light-induced electrical and chemical gradients. Nineteen of 20 commonly occurring amino acids have been shown to be actively accumulated by these vesicles in response to illumination or in response to an artificially created Na+ gradient. On the basis of shared common carriers the transport systems can be divided into eight classes, each responsible for the transport of one or several amino acids: arginine, lysine, histidine; asparagine, glutamine; alanine, glycine, threonine, serine; leucine, valine, isoleucine, methionine; phenylalanine, tyrosine, tryptophan; aspartate; glutamate; proline. Available evidence suggests that these carriers are symmetrical in that amino acids can be transported equally well in both directions across the vesicle membranes. A tentative working model to account for these observations is presented.

  11. Biosynthesis of 'essential' amino acids by scleractinian corals.

    PubMed Central

    Fitzgerald, L M; Szmant, A M

    1997-01-01

    Animals rely on their diet for amino acids that they are incapable either of synthesizing or of synthesizing in sufficient quantities to meet metabolic needs. These are the so-called 'essential amino acids'. This set of amino acids is similar among the vertebrates and many of the invertebrates. Previously, no information was available for amino acid synthesis by the most primitive invertebrates, the Cnidaria. The purpose of this study was to examine amino acid synthesis by representative cnidarians within the Order Scleractinia. Three species of zooxanthellate reef coral, Montastraea faveolata, Acropora cervicornis and Porites divaricata, and two species of non-zooxanthellate coral, Tubastrea coccinea and Astrangia poculata, were incubated with 14C-labelled glucose or with the 14C-labelled amino acids glutamic acid, lysine or valine. Radiolabel tracer was followed into protein amino acids. A total of 17 amino acids, including hydroxyproline, were distinguishable by the techniques used. Of these, only threonine was not found radiolabelled in any of the samples. We could not detect tryptophan or cysteine, nor distinguish between the amino acid pairs glutamic acid and glutamine, or aspartic acid and asparagine. Eight amino acids normally considered essential for animals were made by the five corals tested, although some of them were made only in small quantities. These eight amino acids are valine, isoleucine, leucine, tyrosine, phenylalanine histidine, methionine and lysine. The ability of cnidarians to synthesize these amino acids could be yet another indicator of a separate evolutionary history of the cnidarians from the rest of the Metazoa. PMID:9078264

  12. Acute Administration of Methionine Affects Performance of Swiss Mice in Learning and Memory Paradigms.

    PubMed

    Abi, I; Magaji, R A; Magaji, M G

    2015-12-20

    Methionine, an essential amino acid, plays an essential role in the central nervous system CNS development. It serves as a crucial intermediate in the methylation, trans-sulfuration and amino- phosphorylationpathways,necessary for the synthesis of nucleic acids, phospholipids, hormones, neurotransmitters, antioxidants, polyamines, catecholamines and other biogenic amines. The effect of methionine on learning and memory in mice was investigated using Morris water maze (MWM), Elevated plus maze(EPM) and Y maze (YM). Animals were administered with distilled water (control), methionine (1,700mg/kg); folate (3mg/kg) or methionine (1700mg/kg) plus folate (3mg/kg) for 14 days. Escape latency and time spent in target quadrants; transfer latency and percentage spontaneous alternations were measured in the MWM, EPM and YM respectively. The animals were anaesthetized with inhalational chloroform and their brains subsequently harvested, homogenized and assayed for acetylcholinesterase24 hours after the experiment.Folate significantly(p<0.05) increased transfer latency (53.33 ± 12.62) as compared to control (20.1 ± 5.01) and reduced spontaneous alternations significantly (25.0 ± 8.9) when compared to control (44.33 ± 3.07). When folate was combined with methionine there was also a significant increase in transfer latency (43.0 ± 14.39) when compared with control (20.1 ± 5.01). Folate-methionine combination also significantly reduced spontaneous alternations (20.4 ± 8.4) as compared to the control (44.33 ± 3.07) much more than folate alone. Acetylcholinesterase activities in all groups were not statistically significant. It can be concluded that acute methionine administration has some benefits in memory enhancement. However, a short course folate supplementation impairslearning and working memory especially when combined with methioninewhich may be as a result of sudden overwhelming of the methylation cycle, leading to homocysteinemia which is pro-dementia.

  13. Acute Administration of Methionine Affects Performance of Swiss Mice in Learning and Memory Paradigms.

    PubMed

    Abi, I; Magaji, R A; Magaji, M G

    2015-01-01

    Methionine, an essential amino acid, plays an essential role in the central nervous system CNS development. It serves as a crucial intermediate in the methylation, trans-sulfuration and amino- phosphorylationpathways,necessary for the synthesis of nucleic acids, phospholipids, hormones, neurotransmitters, antioxidants, polyamines, catecholamines and other biogenic amines. The effect of methionine on learning and memory in mice was investigated using Morris water maze (MWM), Elevated plus maze(EPM) and Y maze (YM). Animals were administered with distilled water (control), methionine (1,700mg/kg); folate (3mg/kg) or methionine (1700mg/kg) plus folate (3mg/kg) for 14 days. Escape latency and time spent in target quadrants; transfer latency and percentage spontaneous alternations were measured in the MWM, EPM and YM respectively. The animals were anaesthetized with inhalational chloroform and their brains subsequently harvested, homogenized and assayed for acetylcholinesterase24 hours after the experiment.Folate significantly(p<0.05) increased transfer latency (53.33 ± 12.62) as compared to control (20.1 ± 5.01) and reduced spontaneous alternations significantly (25.0 ± 8.9) when compared to control (44.33 ± 3.07). When folate was combined with methionine there was also a significant increase in transfer latency (43.0 ± 14.39) when compared with control (20.1 ± 5.01). Folate-methionine combination also significantly reduced spontaneous alternations (20.4 ± 8.4) as compared to the control (44.33 ± 3.07) much more than folate alone. Acetylcholinesterase activities in all groups were not statistically significant. It can be concluded that acute methionine administration has some benefits in memory enhancement. However, a short course folate supplementation impairslearning and working memory especially when combined with methioninewhich may be as a result of sudden overwhelming of the methylation cycle, leading to homocysteinemia which is pro-dementia. PMID

  14. Decreased rat rhabdomyosarcoma pulmonary metastases in response to a low methionine diet.

    PubMed

    Breillout, F; Hadida, F; Echinard-Garin, P; Lascaux, V; Poupon, M F

    1987-01-01

    Many Experimental and human tumor cell lines have been previously described as being dependent upon exogenous methionine for their in vitro proliferation. The rationale of the experiments described herein was to decrease the in vivo growth of malignant tumors by reducing the exogenous methionine available in diets fed to Wistar AG rats bearing the highly metastatic rhabdomyosarcoma, RMS-J1. The methionine content in the diet was reduced either by replacing casein (diet 1) with soybean protein (diet 4), or by lowering the amount of soybean protein in the diet (from 23 g/100 g to 12 g/100g) (diet 5), or by using a crystalline amino acid-defined mixture as the source of protein (diet 7). In the latter diet homocysteine replaced methionine and allowed the survival of the animals. Diet 4 significantly reduced the mean number of lung metastases without affecting the primary tumor growth. Treatment of RMS-J1 bearing rats with diet 5 led to the decrease of pulmonary invasion (78 and 21 median lung metastases, respectively, in control and treated groups). This diminished metastatic dissemination resulted from the reduced methionine consumption: the lowered casein content in diet 3 (10 g/100 g) as compared to diet 1 (23 g) did not alter primary tumor growth or the amplitude of lung invasion. Moreover, the addition of methionine to diet 5 prevented the diminution of the median number of lung metastases. Replacement of methionine with homocysteine in the crystalline amino acid-defined mixture (diet 7) fed to RMS-J1 bearing rats led to a limited retardation of primary tumor growth (less than 10%) and to a significant decrease in pulmonary invasion: the median number of pulmonary metastases was 28 and 9 for control and treated rats respectively.

  15. Structural insights into the recovery of aldolase activity in N-acetylneuraminic acid lyase by replacement of the catalytically active lysine with γ-thialysine by using a chemical mutagenesis strategy.

    PubMed

    Timms, Nicole; Windle, Claire L; Polyakova, Anna; Ault, James R; Trinh, Chi H; Pearson, Arwen R; Nelson, Adam; Berry, Alan

    2013-03-01

    Chemical modification has been used to introduce the unnatural amino acid γ-thialysine in place of the catalytically important Lys165 in the enzyme N-acetylneuraminic acid lyase (NAL). The Staphylococcus aureus nanA gene, encoding NAL, was cloned and expressed in E. coli. The protein, purified in high yield, has all the properties expected of a class I NAL. The S. aureus NAL which contains no natural cysteine residues was subjected to site-directed mutagenesis to introduce a cysteine in place of Lys165 in the enzyme active site. Subsequently chemical mutagenesis completely converted the cysteine into γ-thialysine through dehydroalanine (Dha) as demonstrated by ESI-MS. Initial kinetic characterisation showed that the protein containing γ-thialysine regained 17 % of the wild-type activity. To understand the reason for this lower activity, we solved X-ray crystal structures of the wild-type S. aureus NAL, both in the absence of, and in complex with, pyruvate. We also report the structures of the K165C variant, and the K165-γ-thialysine enzyme in the presence, or absence, of pyruvate. These structures reveal that γ-thialysine in NAL is an excellent structural mimic of lysine. Measurement of the pH-activity profile of the thialysine modified enzyme revealed that its pH optimum is shifted from 7.4 to 6.8. At its optimum pH, the thialysine-containing enzyme showed almost 30 % of the activity of the wild-type enzyme at its pH optimum. The lowered activity and altered pH profile of the unnatural amino acid-containing enzyme can be rationalised by imbalances of the ionisation states of residues within the active site when the pK(a) of the residue at position 165 is perturbed by replacement with γ-thialysine. The results reveal the utility of chemical mutagenesis for the modification of enzyme active sites and the exquisite sensitivity of catalysis to the local structural and electrostatic environment in NAL.

  16. Bioavailability of lysine in selected foods by rat growth assay.

    PubMed

    McDonough, F E; Bodwell, C E; Hitchins, A D; Staples, R S

    1989-01-01

    Lysine bioavailabilities in reference protein and 16 test protein diets were estimated using 10 day rat growth assays. A standard growth curve was obtained by feeding 5 diets containing casein, zein and synthetic amino acids ranging in total lysine concentration from 0.3 to 0.7%. Experimental foods were added to the basal diet at the expense of zein and/or synthetic amino acids to provide 2 specific lysine concentrations, i.e., 0.4 and 0.6%. Availabilities were established by comparing growth responses from the test food diets to the regression line of the standard growth data. Availabilities were over 88% for 13 of 16 products. Utilization was poor in pinto beans (73%), rice-wheat gluten cereal (70%), and skim milk powder heated to 100 degrees C for 12 h (66%). Addition of excess lysine (700 mg/100 g diet) to the pinto bean diet did not improve growth response; thus poor digestibility or some unidentified growth inhibitor is indicated. PMID:2496403

  17. Amperometric biosensor based on diamond paste for the enantioanalysis of L-lysine.

    PubMed

    Stefan-van Staden, Raluca-Ioana; Nejem, R'afat Mahmoud; van Staden, Jacobus Frederick; Aboul-Enein, Hassan Y

    2012-05-15

    An amperometric biosensor was proposed for the enantioanalysis of L-lysine. The biosensor is based on the impregnation of L-lysine oxidase in diamond paste. The potential used for the determination of l-lysine was 650 mV. The biosensor exhibited a linear concentration range between 1 and 100 nmol/L with a limit of detection of 4 pmol/L. The selectivity of the biosensor is high over other amino acids, such as L-serine, L-leucine, L-aspartic acid, L-glutamic acid, histamine, glycine. The proposed biosensor can be applied for the determination of L-lysine in serum samples and pharmaceutical compounds.

  18. Conserved methionine dictates substrate preference in Nramp-family divalent metal transporters.

    PubMed

    Bozzi, Aaron T; Bane, Lukas B; Weihofen, Wilhelm A; McCabe, Anne L; Singharoy, Abhishek; Chipot, Christophe J; Schulten, Klaus; Gaudet, Rachelle

    2016-09-13

    Natural resistance-associated macrophage protein (Nramp) family transporters catalyze uptake of essential divalent transition metals like iron and manganese. To discriminate against abundant competitors, the Nramp metal-binding site should favor softer transition metals, which interact either covalently or ionically with coordinating molecules, over hard calcium and magnesium, which interact mainly ionically. The metal-binding site contains an unusual, but conserved, methionine, and its sulfur coordinates transition metal substrates, suggesting a vital role in their transport. Using a bacterial Nramp model system, we show that, surprisingly, this conserved methionine is dispensable for transport of the physiological manganese substrate and similar divalents iron and cobalt, with several small amino acid replacements still enabling robust uptake. Moreover, the methionine sulfur's presence makes the toxic metal cadmium a preferred substrate. However, a methionine-to-alanine substitution enables transport of calcium and magnesium. Thus, the putative evolutionary pressure to maintain the Nramp metal-binding methionine likely exists because it-more effectively than any other amino acid-increases selectivity for low-abundance transition metal transport in the presence of high-abundance divalents like calcium and magnesium. PMID:27573840

  19. Genetic engineering for high methionine grain legumes.

    PubMed

    Müntz, K; Christov, V; Saalbach, G; Saalbach, I; Waddell, D; Pickardt, T; Schieder, O; Wüstenhagen, T

    1998-08-01

    Methionine (Met) is the primary limiting essential amino acid in grain legumes. The imbalance in amino acid composition restricts their biological value (BV) to 55 to 75% of that of animal protein. So far improvement of the BV could not be achieved by conventional breeding. Therefore, genetic engineering was employed by several laboratories to resolve the problem. Three strategies have been followed. A) Engineering for increased free Met levels; B) engineering of endogenous storage proteins with increased numbers of Met residues; C) transfer of foreign genes encoding Met-rich proteins, e.g. the Brazil nut 2S albumin (BNA) and its homologue from sunflower, into grain legumes. The latter strategy turned out to be most promising. In all cases the gene was put under the control of a developmentally regulated seed specific promoter and transferred into grain legumes using the bacterial Agrobacterium tumefaciens-system. Integration into and copy numbers in the plant genome as well as Mendelian inheritance and gene dosage effects were verified. After correct precursor processing the mature 2S albumin was intracellularly deposited in protein bodies which are part of the vacuolar compartment. The foreign protein amounted to 5 to 10% of the total seed protein in the best transgenic lines of narbon bean (Vicia narbonensis L., used in the authors' laboratories), lupins (Lupinus angustifolius L., used in CSIRO, Australia), and soybean (Glycine max (L.) Merr., used by Pioneer Hi-Bred, Inc., USA). In the narbon bean the increase of Met was directly related to the amount of 2S albumin in the transgenic seeds, but in soybean it remained below the theoretically expected value. Nevertheless, trangenic soybean reached 100%, whereas narbon bean and lupins reached approximately 80% of the FAO-standard for nutritionally balanced food proteins. These results document that the Met problem of grain legumes can be resolved by genetic engineering.

  20. Evaluation of Methionine Content in a High-Fat and Choline-Deficient Diet on Body Weight Gain and the Development of Non-Alcoholic Steatohepatitis in Mice

    PubMed Central

    Chiba, Tsuyoshi; Suzuki, Sachina; Sato, Yoko; Itoh, Tatsuki; Umegaki, Keizo

    2016-01-01

    Aim Non-alcoholic steatohepatitis (NASH) is a globally recognized liver disease. A methionine- and choline-deficient diet is used to induce NASH in mice; however, this diet also causes severe body weight loss. To resolve this issue, we examined the effects of methionine content in a high-fat and choline-deficient (HFCD) diet on body weight and the development of NASH in mice. Methods C57BL/6J mice (male, 10 weeks of age) were fed an L-amino acid rodent (control) diet, high-fat (HF) diet, or HFCD diet containing various amounts of methionine (0.1–0.6% (w/w)) for 12 weeks. Plasma lipid levels, hepatic lipid content and inflammatory marker gene expression were measured, and a pathological analysis was conducted to evaluate NASH. Results The 0.1% methionine in HFCD diet suppressed body weight gain, which was lower than that with control diet. On the other hand, the 0.2% methionine in HFCD diet yielded similar body weight gains as the control diet, while more than 0.4% methionine showed the same body weight gains as the HF diet. Liver weights and hepatic lipid contents were the greatest with 0.1% methionine and decreased in a methionine dose-dependent manner. Pathological analysis, NAFLD activity scores and gene expression levels in the liver revealed that 0.1% and 0.2% methionine for 12 weeks induced NASH, whereas 0.4% and 0.6% methionine attenuated the induction of NASH by HFCD diet. However, the 0.2% methionine in HFCD diet did not induce insulin resistance, despite the body weight gain. Conclusions The 0.2% methionine in HFCD diet for 12 weeks was able to induce NASH without weight loss. PMID:27723801

  1. Antitumor effect of methionine-depleting total parenteral nutrition with doxorubicin administration on Yoshida sarcoma-bearing rats.

    PubMed

    Goseki, N; Yamazaki, S; Endo, M; Onodera, T; Kosaki, G; Hibino, Y; Kuwahata, T

    1992-04-01

    Methionine-depleting total parenteral nutrition (methionine-depleting TPN), which infuses an amino acid solution devoid of L-methionine and L-cysteine as the sole protein source, showed enhancement of the effect of several anti-cancer agents. In this study, the combined effect of the methionine-depleting TPN with the administration of doxorubicin was examined in Yoshida sarcoma (YS)-bearing rats with regard to effects on the primary tumor growth, the extension of metastasis, and the host animal's life span. In the first experiment, immediately after receiving methionine-depleting TPN for 8 days, the animals were killed. Pathologic findings evaluated tumor growth in the implanted site and extension of the metastasis. In the second experiment, the survival period was determined after animals received methionine-depleting TPN for 10 days, with subsequent oral feeding until they died naturally. Proliferation of YS was markedly suppressed. In particular, hematogenous metastasis, which is a characteristic of YS, was suppressed, and a longer survival period (42.7 +/- 15.6 days, mean +/- SD) was attained in rats in the group treated with the methionine-depleting TPN combined with the administration of doxorubicin.

  2. Identification, expression and antibacterial activities of an antimicrobial peptide NK-lysin from a marine fish Larimichthys crocea.

    PubMed

    Zhou, Qi-Jia; Wang, Jun; Liu, Min; Qiao, Ying; Hong, Wan-Shu; Su, Yong-Quan; Han, Kun-Huang; Ke, Qiao-Zhen; Zheng, Wei-Qiang

    2016-08-01

    As fundamental immunologic mechanism, the innate immunity system is more important than the specific immunity system in teleost fishes during pathogens infection. Antimicrobial peptides are integral parts of the innate immune system, and play significant roles against pathogens infection. NK-lysin, the compounds of the natural killer cells and cytotoxic T cells, are potent and effective antimicrobial peptides widely distributed in animals. In this study, we reported the sequence characteristics, expression profiles and antibacterial activities of a NK-lysin gene (Lc-NK-lysin) from a commercially important marine fish, the large yellow croaker (Larimichthys crocea). The open reading frame of Lc-NK-lysin cDNA sequence was 447 bp in length, coding 148 amino acids. The genomic DNA of Lc-NK-lysin has the common features of NK-lysin family, consisting of five exons and four introns, and in its deduced mature peptide, there are six well-conserved cysteine residues and a Saposin B domain. Lc-NK-lysin was expressed in all tested tissues (skin, muscle, gill, brain, head kidney, heart, liver, spleen, stomach and intestine) with different expression patterns. In pathogens infection the expression profiles of Lc-NK-lysin varied significantly in gill, head kidney, spleen and liver, indicating its role in immune response. Two peptides (Lc-NK-lysin-1 and Lc-NK-lysin-2) divided from the core region of the Lc-NK-lysin mature polypeptide were chemically synthesized and their antibacterial activities were examined; the potential function on the inhibition of bacteria propagation was revealed. Our results suggested that Lc-NK-lysin is a typical member of the NK-lysin family and as an immune-related gene it involves in the immune response when pathogens invasion. PMID:27238427

  3. The development and amino acid binding ability of nano-materials based on azo derivatives: theory and experiment.

    PubMed

    Shang, Xuefang; Du, Jinge; Yang, Wancai; Liu, Yun; Fu, Zhiyuan; Wei, Xiaofang; Yan, Ruifang; Yao, Ningcong; Guo, Yaping; Zhang, Jinlian; Xu, Xiufang

    2014-05-01

    Two nano-material-containing azo groups have been designed and developed, and the binding ability of nano-materials with various amino acids has been characterized by UV-vis and fluorescence titrations. Results indicated that two nano-materials showed the strongest binding ability for homocysteine among twenty normal kinds of amino acids (alanine, valine, leucine, isoleucine, methionine, aspartic acid, glutamic acid, arginine, glycine, serine, threonine, asparagine, phenylalanine, histidine, tryptophan, proline, lysine, glutamine, tyrosine and homocysteine). The reason for the high sensitivity for homocysteine was that two nano-materials containing an aldehyde group reacted with SH in homocysteine and afforded very stable thiazolidine derivatives. Theoretical investigation further illustrated the possible binding mode in host-guest interaction and the roles of molecular frontier orbitals in molecular interplay. Thus, the two nano-materials can be used as optical sensors for the detection of homocysteine.

  4. The importance of transmethylation reactions to methionine metabolism in sheep: effects of supplementation with creatine and choline.

    PubMed

    Lobley, G E; Connell, A; Revell, D

    1996-01-01

    The influence of administering the methylated products choline and creatine on methionine irreversible-loss rate (ILR) and recycling from homocysteine has been investigated in sheep fed close to energy and N equilibrium. Two methods to estimate methionine recycling were compared. The first involved [U-13C]methionine infused as part of a labelled amino acid mixture obtained from hydrolysed algal protein. In this approach the isotope dilution of methionine with all five C atoms labelled (m + 5) will represent the ILR which does not recycle through homocysteine, while that which includes molecules with C-1-C-4 labelled will allow for loss of the labelled methyl (5)-C atom and replacement by an unlabelled moiety in the remethylation of homocysteine. The second method involved a combined infusion of [1-13C]- and [S-methyl-2H3]methionine. These two approaches gave similar data for methionine ILR which does not include label recycled to the amino acid from homocysteine but differed for recycled methionine fluxes. Consequently the two procedures differed in the calculated extent of homocysteine methylation under control conditions (6 v. 28%). These extents of remethylation are within the range observed for the fed human subject, despite the fact that fewer dietary methyl groups are available for the ruminant. Using combined data from the infusions, significant depression of methionine recycling occurred in blood (P < 0.05), with a similar trend for plasma (P = 0.077), when choline plus creatine were infused. Wool growth, assessed by intradermal injection of [35S]cysteine, was not altered by supplementation with the methylated products. From changes in the label pattern of free methionine in aortal, hepatic portal and hepatic venous blood during U-13C-labelled algal hydrolysate infusion, the major sites of homocysteine remethylation appear to be the portal-drained viscera and the liver. This was confirmed by analysis of free methionine enrichments in various tissues

  5. Investigation of Lysine-Functionalized Dendrimers as Dichlorvos Detoxification Agents.

    PubMed

    Durán-Lara, Esteban F; Marple, Jennifer L; Giesen, Joseph A; Fang, Yunlan; Jordan, Jacobs H; Godbey, W Terrence; Marican, Adolfo; Santos, Leonardo S; Grayson, Scott M

    2015-11-01

    Lysine-containing polymers have seen broad application due to their amines' inherent ability to bind to a range of biologically relevant molecules. The synthesis of multiple generations of polyester dendrimers bearing lysine groups on their periphery is described in this report. Their hydrolytic stabilities with respect to pH and time, their toxicity to a range of cell lines, and their possible application as nano-detoxification agents of organophosphate compounds are all investigated. These zeroth-, first-, and second-generation water-soluble dendrimers have been designed to bear exactly 4, 8, and 16 lysine groups, respectively, on their dendritic periphery. Such monodisperse bioactive polymers show potential for a range of applications including drug delivery, gene delivery, heavy metal binding, and the sequestration of organic toxins. These monodisperse bioactive dendrimers were synthesized using an aliphatic ester dendritic core (prepared from pentaerythritol) and protected amino acid moieties. This library of lysine-conjugated dendrimers showed the ability to efficiently capture the pesticide dichlorvos, confirming the potential of dendrimer-based antidotes to maintain acetylcholinesterase activity in response to poisoning events. PMID:26460283

  6. Protein lysine methylation by seven-β-strand methyltransferases.

    PubMed

    Falnes, Pål Ø; Jakobsson, Magnus E; Davydova, Erna; Ho, Angela; Małecki, Jędrzej

    2016-07-15

    Methylation of biomolecules is a frequent biochemical reaction within the cell, and a plethora of highly specific methyltransferases (MTases) catalyse the transfer of a methyl group from S-adenosylmethionine (AdoMet) to various substrates. The posttranslational methylation of lysine residues, catalysed by numerous lysine (K)-specific protein MTases (KMTs), is a very common and important protein modification, which recently has been subject to intense studies, particularly in the case of histone proteins. The majority of KMTs belong to a class of MTases that share a defining 'SET domain', and these enzymes mostly target lysines in the flexible tails of histones. However, the so-called seven-β-strand (7BS) MTases, characterized by a twisted beta-sheet structure and certain conserved sequence motifs, represent the largest MTase class, and these enzymes methylate a wide range of substrates, including small metabolites, lipids, nucleic acids and proteins. Until recently, the histone-specific Dot1/DOT1L was the only identified eukaryotic 7BS KMT. However, a number of novel 7BS KMTs have now been discovered, and, in particular, several recently characterized human and yeast members of MTase family 16 (MTF16) have been found to methylate lysines in non-histone proteins. Here, we review the status and recent progress on the 7BS KMTs, and discuss these enzymes at the levels of sequence/structure, catalytic mechanism, substrate recognition and biological significance. PMID:27407169

  7. Increased synthesis of eicosanoids by human monocytes following leucine and methionine enkephalin administration

    SciTech Connect

    Wiederhold, M.D.; Ou, D.W.

    1986-03-05

    Regulation of eicosanoid biosynthesis by neuropeptides was investigated in human peripheral blood monocytes from normal donors. Metabolites of /sup 3/H-arachidonic acid (/sup 3/H-AA) were analyzed by thin layer and high pressure liquid chromatography following exposure to 0.2 ..mu..gm/ml and 2.0 ..mu..gm/ml of leucine (L-ENK) and methionine (M-ENK) enkephalin. Supernatants of cultured cells were analyzed. The data indicate that both leucine and methionine enkephalin can stimulate eicosanoid biosynthesis in human monocytes, and may indicate a possible regulatory mechanism between the central nervous system and the reticuloendothelial system.

  8. Isotopic Dilution GC/MS Method for Methionine Determination in Biological Media

    NASA Astrophysics Data System (ADS)

    Horj, Elena; Iordache, Andreea; Culea, Monica

    2011-10-01

    The isotopic dilution mass spectrometry technique is the method of choice for sensitive and accurate determination of analytes in biological samples. The aim of this work was to establish a sensitive analytical method for the determination of methionine in different biological media. Quantitation of methionine from the resultant tracer spectrum requires deconvolution of the enrichment of the isotopomers. Deconvolution of the ion abundance ratios to yield tracer-to-tracee ratio for the isotopomer was done using Brauman's least squares approach. Comparison with regression curve calculation method is presented. The method was applied for amino-acids determination in beef, pork and fish meat.

  9. Coacervate-like microspheres from lysine-rich proteinoid

    NASA Technical Reports Server (NTRS)

    Rohlfing, D. L.

    1975-01-01

    Microspheres form isothermally from lysine-rich proteinoid when the ionic strength of the solution is increased with NaCl or other salts. Studies with different monovalent anions and with polymers of different amino acid composition indicate that charge neutralization and hydrophobic bonding contribute to microsphere formation. The particles also form in sea water, especially if heated or made slightly alkaline. The microspheres differ from those made from acidic proteinoid but resemble coacervate droplets in some ways (isothermal formation, limited stability, stabilization by quinone, uptake of dyes). Because the constituent lysine-rich proteinoid is of simulated prebiotic origin, the study is interpreted to add emphasis to and suggest an evolutionary continuity for coacervation phenomena.

  10. Parasites suppress immune-enhancing effect of methionine in nestling great tits.

    PubMed

    Wegmann, Michèle; Voegeli, Beatrice; Richner, Heinz

    2015-01-01

    After birth, an organism needs to invest both in somatic growth and in the development of efficient immune functions to counter the effects of pathogens, and hence an investment trade-off is predicted. To explore this trade-off, we simultaneously exposed nestling great tits (Parus major) to a common ectoparasite, while stimulating immune function. Using a 2 × 2 experimental design, we first infested half of the nests with hen fleas (Ceratophyllus gallinae) on day 3 post-hatch and later, on day 9-13 post-hatch, and then supplemented half of the nestlings within each nest with an immuno-enhancing amino acid (methionine). We then assessed the non-specific immune response by measuring both the inflammatory response to a lipopolysaccharide (LPS) and assessing the levels of acute phase proteins (APP). In parasite-infested nestlings, methionine had a negative effect on body mass close to fledging. Methionine had an immune-enhancing effect in the absence of ectoparasites only. The inflammatory response to LPS was significantly lower in nestlings infested with fleas and was also lower in nestlings supplemented with methionine. These patterns of immune responses suggest an immunosuppressive effect of ectoparasites that could neutralise the immune-enhancing effect of methionine. Our study thus suggests that the trade-off between investment in life history traits and immune function is only partly dependent on available resources, but shows that parasites may influence this trade-off in a more complex way, by also inhibiting important physiological functions.

  11. Suppression of Methionine Oxidation of a Pharmaceutical Antibody Stored in a Polymer-Based Syringe.

    PubMed

    Masato, Amano; Kiichi, Fukui; Uchiyama, Susumu

    2016-02-01

    Oxidation of methionine residues is one of the well-known deteriorations in monoclonal antibody (mAb) therapeutics. Because methionine oxidation may affect their efficacy and pharmacokinetic profile, oxidation levels should be strictly controlled during their storage period. In this study, we revealed that when a therapeutic antibody was filled into a cyclo olefin polymer-based syringe and stored in a blister pack with an oxygen absorber, the methionine oxidation production under thermal or light stress was suppressed because of the reduction in the concentration of dissolved oxygen. Also unexpectedly, fewer amounts of the high-molecular-weight species and the acidic variants of the antibody were generated under thermal or light stress. Although the high-molecular-weight species contains methionine oxidants at similar levels to those in a monomer species, they were likely to be constituted from a higher amount of the oxidative species of internal disulfide linkage, tyrosine, or histidine. Because the dissolved oxygen could be readily removed from the mAb solution in the polymer-based syringe owing to its high gas permeability, this study shows the advantages of the polymer-based syringe with an oxygen absorber over glass syringes in terms of the suppression of the methionine oxidation and oxidative high molecular species. PMID:26462145

  12. A Methionine-Induced Animal Model of Schizophrenia: Face and Predictive Validity

    PubMed Central

    Wang, Lien; Alachkar, Amal; Sanathara, Nayna; Belluzzi, James D.; Wang, Zhiwei

    2015-01-01

    Background: Modulating the methylation process induces broad biochemical changes, some of which may be involved in schizophrenia. Methylation is in particular central to epigenesis, which is also recognized as a factor in the etiology of schizophrenia. Because methionine administration to patients with schizophrenia has been reported to exacerbate their psychotic symptoms and because mice treated with methionine exhibited social deficits and prepulse inhibition impairment, we investigated whether methionine administration could lead to behavioral changes that reflect schizophrenic symptoms in mice. Methods: l-Methionine was administered to mice twice a day for 7 days. Results: We found that this treatment induces behavioral responses that reflect the 3 types of schizophrenia-like symptoms (positive, negative, or cognitive deficits) as monitored in a battery of behavioral assays (locomotion, stereotypy, social interaction, forced swimming, prepulse inhibition, novel object recognition, and inhibitory avoidance). Moreover, these responses were differentially reversed by typical haloperidol and atypical clozapine antipsychotics in ways that parallel their effects in schizophrenics. Conclusion: We thus propose the l-methionine treatment as an animal model recapitulating several symptoms of schizophrenia. We have established the face and predictive validity for this model. Our model relies on an essential natural amino acid and on an intervention that is relatively simple and time effective and may offer an additional tool for assessing novel antipsychotics. PMID:25991655

  13. Utilization of supplemental methionine sources by primary cultures of chick hepatocytes

    SciTech Connect

    Dibner, J.J.

    1983-10-01

    Utilization of 2-hydroxy-4-(methylthio) butanoic acid (HMB) as a substrate for protein synthesis was studied by using primary cultures of chick liver cells. Cultures were prepared by enzymatic dissociation of livers from week old Hubbard broiler chicks and were maintained for 4 days under nonproliferative conditions. Hepatocyte differentiation was verified by using dexamethasone induction of tyrosine aminotransferase activity. Conversion of (14C)HMB to L-methionine was shown by chromatographic analysis of hepatocyte protein hydrolysate and incorporation into protein was proven by cycloheximide inhibition of synthesis. When incorporation of HMB was compared to that of DL-methionine (DLM) equimolar quantities of the two sources were found in liver cell protein. These results support, at a cellular level, the conclusion that HMB and DLM are biochemically equivalent sources of methionine for protein synthesis.

  14. The use of Streptococcus zymogenes for estimating tryptophan and methionine bioavailability in 17 foods.

    PubMed

    Wells, P; McDonough, F; Bodwell, C E; Hitchens, A

    1989-01-01

    As part of a cooperative study assessing amino acid bioavailability and/or protein quality, the provisional method of Boyne et al. (Brit J Nutr 21: 181-206) was used to assay 17 protein sources for methionine and tryptophan availability with S. zymogenes. Pronase was used as the predigesting enzyme. Product composition was found to affect reproducibility. The microbial assay results correlated positively with results from rat growth studies on the same foods (p = 0.05), and were generally accurate in identifying products of lower protein quality. Defatting four high-fat products increased microbial values in the methionine assay, but only the chicken franks and the sausage values in the tryptophan assay. Heating non-fat milk increased methionine values slightly. Low values for rolled oats were further reduced by finer grinding.

  15. Identification and characterization of lysine-methylated sites on histones and non-histone proteins.

    PubMed

    Lee, Tzong-Yi; Chang, Cheng-Wei; Lu, Cheng-Tzung; Cheng, Tzu-Hsiu; Chang, Tzu-Hao

    2014-06-01

    Protein methylation is a kind of post-translational modification (PTM), and typically takes place on lysine and arginine amino acid residues. Protein methylation is involved in many important biological processes, and most recent studies focused on lysine methylation of histones due to its critical roles in regulating transcriptional repression and activation. Histones possess highly conserved sequences and are homologous in most species. However, there is much less sequence conservation among non-histone proteins. Therefore, mechanisms for identifying lysine-methylated sites may greatly differ between histones and non-histone proteins. Nevertheless, this point of view was not considered in previous studies. Here we constructed two support vector machine (SVM) models by using lysine-methylated data from histones and non-histone proteins for predictions of lysine-methylated sites. Numerous features, such as the amino acid composition (AAC) and accessible surface area (ASA), were used in the SVM models, and the predictive performance was evaluated using five-fold cross-validations. For histones, the predictive sensitivity was 85.62% and specificity was 80.32%. For non-histone proteins, the predictive sensitivity was 69.1% and specificity was 88.72%. Results showed that our model significantly improved the predictive accuracy of histones compared to previous approaches. In addition, features of the flanking region of lysine-methylated sites on histones and non-histone proteins were also characterized and are discussed. A gene ontology functional analysis of lysine-methylated proteins and correlations of lysine-methylated sites with other PTMs in histones were also analyzed in detail. Finally, a web server, MethyK, was constructed to identify lysine-methylated sites. MethK now is available at http://csb.cse.yzu.edu.tw/MethK/.

  16. Segregation for endosperm lysine in F2, F 3 and F 4 progeny from a cross of in vitro-selected and unselected cultivar of rice.

    PubMed

    Schaeffer, G W; Sharpe, F T; Dudley, J T

    1989-02-01

    Lysine is a limiting amino acid for optimal nutritional quality in rice grain. In vitro selections using inhibitory levels of lysine plus threonine or s-aminoethylcysteine allow the predictable recovery of variants with elevated levels of lysine and protein. These methods may generate useful starting germplasm for plant breeders. This study was conducted to define the genetics of lysine mutants in progeny from crosses of mutants derived from cells cultured in vitro in the presence of inhibitory levels of lysine plus threonine and s-(2-aminoethyl)-cysteine. In vitro selections produce a wide range of mutants, including endosperm mutants with elevated lysine and protein levels as well as mutants for high and low seed weights. Mutants were analyzed for lysine content by the endosperm half-seed method in which the halves without the embryo were ground and acid hydrolyzed for amino acid determinations. The halves with the embryos were preserved for later germination. In two different F2 populations derived from a cross of a selected mutant x M-101, a parental marker, there was an inverse relationship between seed weight and percent lysine in endosperm protein (R(2) 0.52 and 0.56). The F2 segregation patterns show that elevated lysine is inherited as a recessive gene and that increased lysine is correlated with decreased seed size. F3 and F4 data provide evidence for the transmission of high lysine genes to advanced germplasm in rice. This work supports our earlier conclusions that high lysine phenotypes can be recovered predictably from in vitro selections. The elevated lysine phenotypes are frequently, but not exclusively, associated with opaque seed. Some segregants from crosses produced increased lysine in plants with near normal seed weight and good fertility. PMID:24232525

  17. Characterization of methionine oxidation and methionine sulfoxide reduction using methionine-rich cysteine-free proteins

    PubMed Central

    2012-01-01

    Background Methionine (Met) residues in proteins can be readily oxidized by reactive oxygen species to Met sulfoxide (MetO). MetO is a promising physiological marker of oxidative stress and its inefficient repair by MetO reductases (Msrs) has been linked to neurodegeneration and aging. Conventional methods of assaying MetO formation and reduction rely on chromatographic or mass spectrometry procedures, but the use of Met-rich proteins (MRPs) may offer a more streamlined alternative. Results We carried out a computational search of completely sequenced genomes for MRPs deficient in cysteine (Cys) residues and identified several proteins containing 20% or more Met residues. We used these MRPs to examine Met oxidation and MetO reduction by in-gel shift assays and immunoblot assays with antibodies generated against various oxidized MRPs. The oxidation of Cys-free MRPs by hydrogen peroxide could be conveniently monitored by SDS-PAGE and was specific for Met, as evidenced by quantitative reduction of these proteins with Msrs in DTT- and thioredoxin-dependent assays. We found that hypochlorite was especially efficient in oxidizing MRPs. Finally, we further developed a procedure wherein antibodies made against oxidized MRPs were isolated on affinity resins containing same or other oxidized or reduced MRPs. This procedure yielded reagents specific for MetO in these proteins, but proved to be ineffective in developing antibodies with broad MetO specificity. Conclusion Our data show that MRPs provide a convenient tool for characterization of Met oxidation, MetO reduction and Msr activities, and could be used for various aspects of redox biology involving reversible Met oxidation. PMID:23088625

  18. Intestinal transport of monosaccharides and amino acids during postnatal development of mink.

    PubMed

    Buddington, R K; Malo, C; Sangild, P T; Elnif, J

    2000-12-01

    Intestinal development is typically studied using omnivores. For comparative purposes, we examined an altricial carnivore, the mink (Mustela vison). In mink, intestinal dimensions increase up to 8 wk after birth and then remain constant (length) or decrease (mass) into maturity despite continuing gains in body mass. Rates of glucose and fructose transport decline after birth for intact tissues but increase for brush-border membrane vesicles (BBMV). Rates of absorption for five amino acids that are substrates for the acidic (aspartate), basic (lysine), neutral (leucine and methionine), and imino acid (proline) carriers increase between birth and 24 h for intact tissues before declining, but increase after 2 wk for BBMV. The proportion of BBMV amino acid uptake that is Na(+)-dependent increases during development but for aspartate is nearly 100% at all ages. Tracer uptake by BBMV can be inhibited by 100 mmol/l of unlabeled amino acid, except for lysine. BBMV uptake of the dipeptide glycyl-sarcosine does not differ between ages, is not Na(+) dependent, and is only partially inhibited by 100 mmol/l unlabeled dipeptide. Despite the ability to rapidly and efficiently digest high dietary loads of protein, rates of amino acid and peptide absorption are not markedly higher than those of other mammals.

  19. CPLM: a database of protein lysine modifications

    PubMed Central

    Liu, Zexian; Wang, Yongbo; Gao, Tianshun; Pan, Zhicheng; Cheng, Han; Yang, Qing; Cheng, Zhongyi; Guo, Anyuan; Ren, Jian; Xue, Yu

    2014-01-01

    We reported an integrated database of Compendium of Protein Lysine Modifications (CPLM; http://cplm.biocuckoo.org) for protein lysine modifications (PLMs), which occur at active ε-amino groups of specific lysine residues in proteins and are critical for orchestrating various biological processes. The CPLM database was updated from our previously developed database of Compendium of Protein Lysine Acetylation (CPLA), which contained 7151 lysine acetylation sites in 3311 proteins. Here, we manually collected experimentally identified substrates and sites for 12 types of PLMs, including acetylation, ubiquitination, sumoylation, methylation, butyrylation, crotonylation, glycation, malonylation, phosphoglycerylation, propionylation, succinylation and pupylation. In total, the CPLM database contained 203 972 modification events on 189 919 modified lysines in 45 748 proteins for 122 species. With the dataset, we totally identified 76 types of co-occurrences of various PLMs on the same lysine residues, and the most abundant PLM crosstalk is between acetylation and ubiquitination. Up to 53.5% of acetylation and 33.1% of ubiquitination events co-occur at 10 746 lysine sites. Thus, the various PLM crosstalks suggested that a considerable proportion of lysines were competitively and dynamically regulated in a complicated manner. Taken together, the CPLM database can serve as a useful resource for further research of PLMs. PMID:24214993

  20. AMINO ACIDS AND HEMOGLOBIN PRODUCTION IN ANEMIA

    PubMed Central

    Whipple, G. H.; Robscheit-Robbins, F. S.

    1940-01-01

    Certain individual amino acids when given to standard anemic dogs cause an increase in new hemoglobin production. Occasional negative experiments are recorded. Glycine, glutamic acid, aspartic acid, cystine, histidine, phenylalanine, and proline when given in 1 gm. doses daily for 2 weeks, increase hemoglobin output on the average 23 to 25 gm. above the control level. This reaction amounts to 25 to 30 per cent of the new hemoglobin produced by the feeding of 300 gm. liver daily for 2 weeks—a standard liver test. Alanine, valine, isoleucine, and arginine in the same dosage increase the hemoglobin output on the average 13 to 17 gm. per 2 weeks over the control level. Leucine, methionine, lysine, tryptophane, and tyrosine fall in a middle group with hemoglobin output of about 20 gm. Isovaleric acid, β-hydroxybutyric acid, glutaric acid, and asparagine have shown positive effects and the butyrate is unusually potent for hemoglobin production (Table 2). The isomeric and dl-synthetic forms of the amino acids are as effectively utilized in this reaction as are the natural forms. PMID:19870982

  1. Hemoglobin Labeled by Radioactive Lysine

    DOE R&D Accomplishments Database

    Bale, W. F.; Yuile, C. L.; DeLaVergne, L.; Miller, L. L.; Whipple, G. H.

    1949-12-08

    This paper reports on the utilization of tagged epsilon carbon of DL-lysine by a dog both anemic and hypoproteinemic due to repeated bleeding plus a diet low in protein. The experiment extended over period of 234 days, a time sufficient to indicate an erythrocyte life span of at least 115 days based upon the rate of replacement of labeled red cell proteins. The proteins of broken down red cells seem not to be used with any great preference for the synthesis of new hemoglobin.

  2. Toward an era of utilizing methionine overproducing hosts for recombinant protein production in Escherichia coli.

    PubMed

    Veeravalli, Karthik; Laird, Michael W

    2015-01-01

    Amino acid sequence variants, especially variants containing non-canonical amino acids such as norleucine and norvaline, are a concern during therapeutic protein production in microbial systems. Substitution of methionine residues with norleucine in recombinant proteins produced in Escherichia coli is well known. Continuous feeding of amino acids such as methionine is commonly used in E. coli fermentation processes to control incorporation of norleucine in the recombinant protein. There are several disadvantages associated with continuous feeding during a fermentation process. For example, a continuous feed increases the operational complexity and cost of a manufacturing process and results in dilution of culture medium which could result in lower cell densities and product yields. To overcome the limitations of existing approaches to prevent norleucine incorporation during E. coli fermentations, a new approach using an engineered host was developed that overproduces methionine in the cell to prevent norleucine incorporation without negatively impacting fermentation process performance and product yields. In this commentary, the results on using methionine overproducing hosts for recombinant protein production in E. coli and some "watch outs" when using these hosts for recombinant protein production are discussed.

  3. Toward an era of utilizing methionine overproducing hosts for recombinant protein production in Escherichia coli

    PubMed Central

    Veeravalli, Karthik; Laird, Michael W

    2015-01-01

    Amino acid sequence variants, especially variants containing non-canonical amino acids such as norleucine and norvaline, are a concern during therapeutic protein production in microbial systems. Substitution of methionine residues with norleucine in recombinant proteins produced in Escherichia coli is well known. Continuous feeding of amino acids such as methionine is commonly used in E. coli fermentation processes to control incorporation of norleucine in the recombinant protein. There are several disadvantages associated with continuous feeding during a fermentation process. For example, a continuous feed increases the operational complexity and cost of a manufacturing process and results in dilution of culture medium which could result in lower cell densities and product yields. To overcome the limitations of existing approaches to prevent norleucine incorporation during E. coli fermentations, a new approach using an engineered host was developed that overproduces methionine in the cell to prevent norleucine incorporation without negatively impacting fermentation process performance and product yields. In this commentary, the results on using methionine overproducing hosts for recombinant protein production in E. coli and some “watch outs” when using these hosts for recombinant protein production are discussed. PMID:25801611

  4. Rumen degradation and availability of various amounts of liquid methionine hydroxy analog in lactating dairy cows.

    PubMed

    Koenig, K M; Rode, L M; Knight, C D; Vázquez-Añón, M

    2002-04-01

    Ruminal escape of various amounts of methionine hydroxy analog [D,L-2-hydroxy-4-(methylthio)-butanoic acid (HMB)] was measured in an experiment designed as a 4 x 4 Latin square using four lactating dairy cows with cannula in the rumen and duodenum. The cows were fed a diet composed of corn silage, alfalfa haylage, rolled barley grain, canola meal, and blood meal, three times per day. The cows were fed the liquid analog each day for 1 wk before the experiment was started. On the day of the experiment, each cow received an intraruminal bolus dose of 0, 25, or 50 g of the liquid analog (Alimet feed supplement, 88% HMB) or 51.2 g of a dry calcium salt of the analog (86% HMB; MHA) mixed with 0.5 kg of ground barley grain. A liquid phase marker (Co-EDTA) was administered as a bolus dose into the rumen at the time of administration of the methionine hydroxy analogs. Rumen and duodenal contents, and blood serum were collected at 0, 1, 3, 6, 9, 12, and 24 h relative to the time of dosing. Rumen and duodenal samples were analyzed for Co and HMB, and serum was analyzed for free methionine. Fractional rate constants for the passage of the liquid marker (k(p)) and the decline of HMB concentration in the rumen (k(rHMB)) were determined by nonlinear regression. Liquid passage from the rumen was similar among the four analog treatments (0.136 +/- 0.012/h; mean +/- SEM). Ruminal escape of HMB as a percentage of the dose (100% x k(p)/k(rHMB)) did not differ between cows receiving 25, 50, and 51.2 g of the methionine analogs (42.5, 41.0, and 34.9 +/- 9.0%, respectively) and averaged 39.5%. Duodenal appearance of HMB as a percentage also did not differ between cows receiving 25, 50, and 51.2 g of the methionine analogs (16.2, 26.8, and 22.7%, respectively) and averaged 22%. Omasal absorption of HMB was variable ranging from 12.3 to 26.3% and averaged 17.6%. Serum methionine concentration peaked at 3 and 6 h after dosing and increased in proportion to the amount of the analog

  5. Spectrophotometric assays for L-lysine alpha-oxidase and gamma-glutamylamine cyclotransferase.

    PubMed

    Danson, Jedidah W; Trawick, Mary Lynn; Cooper, Arthur J L

    2002-04-15

    A new assay for l-lysine alpha-oxidase is described. In this assay, the oxidized product generated from l-lysine is reacted with semicarbazide to form alpha-keto-epsilon-aminocaproate semicarbazone. Formation of the alpha-keto acid semicarbazone is continuously monitored spectrophotometrically at 248 nm (epsilon 10,160 +/- 240 M(-1) cm(-1)). The method was adapted to provide a new assay for gamma-glutamylamine cyclotransferase. This enzyme catalyzes the conversion of many l-gamma-glutamylamines to 5-oxo-l-proline and free amine. A biologically important substrate is N(epsilon)-(gamma-l-glutamyl)-l-lysine, which is converted to 5-oxo-l-proline and l-lysine by the action of gamma-glutamylamine cyclotransferase. The l-lysine generated from N(epsilon)-(gamma-l-glutamyl)-l-lysine in an endpoint assay is converted to alpha-keto epsilon-aminocaproate semicarbazone in the presence of semicarbazide, excess l-lysine alpha-oxidase, and catalase. The methods were applied to the determination of gamma-glutamylamine cyclotransferase activity of partially purified preparations of the bovine kidney enzyme and to detect gamma-glutamylamine cyclotransferase activity in rat kidney and liver homogenates. PMID:11950211

  6. Structural Basis for l-Lysine Feedback Inhibition of Homocitrate Synthase

    SciTech Connect

    Bulfer, Stacie L.; Scott, Erin M.; Pillus, Lorraine; Trievel, Raymond C.

    2010-09-02

    The {alpha}-aminoadipate pathway of lysine biosynthesis is modulated at the transcriptional and biochemical levels by feedback inhibition. The first enzyme in the {alpha}-aminoadipate pathway, homocitrate synthase (HCS), is the target of the feedback regulation and is strongly inhibited by L-lysine. Here we report the structure of Schizosaccharomyces pombe HCS (SpHCS) in complex with L-lysine. The structure illustrates that the amino acid directly competes with the substrate 2-oxoglutarate for binding within the active site of HCS. Differential recognition of the substrate and inhibitor is achieved via a switch position within the ({alpha}/{beta}){sub 8} TIM barrel of the enzyme that can distinguish between the C5-carboxylate group of 2-oxoglutarate and the {epsilon}-ammonium group of L-lysine. In vitro and in vivo assays demonstrate that mutations of the switch residues, which interact with the L-lysine {epsilon}-ammonium group, abrogate feedback inhibition, as do substitutions of residues within the C-terminal domain that were identified in a previous study of L-lysine-insensitive HCS mutants in Saccharomyces cerevisiae. Together, these results yield new insights into the mechanism of feedback regulation of an enzyme central to lysine biosynthesis.

  7. Genome-Wide Analysis of the Lysine Biosynthesis Pathway Network during Maize Seed Development

    PubMed Central

    Liu, Yuwei; Xie, Shaojun; Yu, Jingjuan

    2016-01-01

    Lysine is one of the most limiting essential amino acids for humans and livestock. The nutritional value of maize (Zea mays L.) is reduced by its poor lysine content. To better understand the lysine biosynthesis pathway in maize seed, we conducted a genome-wide analysis of the genes involved in lysine biosynthesis. We identified lysine biosynthesis pathway genes (LBPGs) and investigated whether a diaminopimelate pathway variant exists in maize. We analyzed two genes encoding the key enzyme dihydrodipicolinate synthase, and determined that they contribute differently to lysine synthesis during maize seed development. A coexpression network of LBPGs was constructed using RNA-sequencing data from 21 developmental stages of B73 maize seed. We found a large set of genes encoding ribosomal proteins, elongation factors and zein proteins that were coexpressed with LBPGs. The coexpressed genes were enriched in cellular metabolism terms and protein related terms. A phylogenetic analysis of the LBPGs from different plant species revealed different relationships. Additionally, six transcription factor (TF) families containing 13 TFs were identified as the Hub TFs of the LBPGs modules. Several expression quantitative trait loci of LBPGs were also identified. Our results should help to elucidate the lysine biosynthesis pathway network in maize seed. PMID:26829553

  8. 21 CFR 172.399 - Zinc methionine sulfate.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Zinc methionine sulfate. 172.399 Section 172.399... CONSUMPTION Special Dietary and Nutritional Additives § 172.399 Zinc methionine sulfate. Zinc methionine... conditions: (a) The additive is the product of the reaction between equimolar amounts of zinc sulfate and...

  9. 21 CFR 172.399 - Zinc methionine sulfate.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Zinc methionine sulfate. 172.399 Section 172.399... CONSUMPTION Special Dietary and Nutritional Additives § 172.399 Zinc methionine sulfate. Zinc methionine... conditions: (a) The additive is the product of the reaction between equimolar amounts of zinc sulfate and...

  10. 21 CFR 172.399 - Zinc methionine sulfate.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Zinc methionine sulfate. 172.399 Section 172.399... CONSUMPTION Special Dietary and Nutritional Additives § 172.399 Zinc methionine sulfate. Zinc methionine... conditions: (a) The additive is the product of the reaction between equimolar amounts of zinc sulfate and...

  11. 21 CFR 172.399 - Zinc methionine sulfate.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Zinc methionine sulfate. 172.399 Section 172.399... CONSUMPTION Special Dietary and Nutritional Additives § 172.399 Zinc methionine sulfate. Zinc methionine... conditions: (a) The additive is the product of the reaction between equimolar amounts of zinc sulfate and...

  12. Effect of different postharvest temperatures on the accumulation of sugars, organic acids, and amino acids in the juice sacs of Satsuma mandarin (Citrus unshiu Marc.) fruit.

    PubMed

    Matsumoto, Hikaru; Ikoma, Yoshinori

    2012-10-01

    To elucidate the effect of different postharvest temperatures on the accumulation of sugars, organic acids, and amino acids and to determine the best temperature to minimize their postharvest change, their content after harvest was investigated at 5, 10, 20, and 30 °C for 14 days in the juice sacs of Satsuma mandarin (Citrus unshiu Marc. cv. Aoshima-unshiu) fruit. In all sugars, the changes were negligible at all temperatures. Organic acids decreased slightly at all temperatures, with the exception of malic acid at 30 °C, which increased slightly. Two amino acids, ornithine and glutamine, increased at 5 °C, but they did not increase at other temperatures. In 11 amino acids (phenylalanine, tryptophan, tyrosine, isoleucine, leucine, valine, threonine, lysine, methionine, histidine, and γ-amino butyric acid), the content was higher at 20 and 30 °C than at other temperatures. Thus, the content of amino acids was more variable than that of sugars and organic acids in response to temperatures. Moreover, amino acids responded to temperature differently: two amino acids were cold responsive, and 11 were heat-responsive. The best temperature to minimize the postharvest changes in amino acid profiles in the juice sacs of Aoshima-unshiu was 10 °C. The responsiveness to temperatures in two cold-responsive (ornithine and glutamine) and five heat-responsive (phenylalanine, tryptophan, valine, lysine, and histidine) amino acids was conserved among three different Satsuma mandarin cultivars, Aoshima-unshiu (late-maturing cultivar), Silverhill (midmaturing cultivar), and Miyagawa-wase (early-maturing cultivar). The metabolic responsiveness to temperature stress was discussed on the basis of the changes in the amino acid profile.

  13. Effective atomic numbers and electron densities of bacteriorhodopsin and its comprising amino acids in the energy range 1 keV-100 GeV

    NASA Astrophysics Data System (ADS)

    Ahmadi, Morteza; Lunscher, Nolan; Yeow, John T. W.

    2013-04-01

    Recently, there has been an interest in fabrication of X-ray sensors based on bacteriorhodopsin, a proton pump protein in cell membrane of Halobacterium salinarium. Therefore, a better understanding of interaction of X-ray photons with bacteriorhodopsin is required. We use WinXCom program to calculate the mass attenuation coefficient of bacteriorhodopsin and its comprising amino acids for photon energies from 1 keV to 100 GeV. These amino acids include alanine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, Asx1, Asx2, Glx1 and Glx2. We then use that data to calculate effective atomic number and electron densities for the same range of energy. We also emphasize on two ranges of energies (10-200 keV and 1-20 MeV) in which X-ray imaging and radiotherapy machines work.

  14. Removal of N-terminal methionine from recombinant proteins by engineered E. coli methionine aminopeptidase

    PubMed Central

    Liao, You-Di; Jeng, Jen-Chong; Wang, Chiu-Feng; Wang, Sui-Chi; Chang, Shu-Ting

    2004-01-01

    The removal of N-terminal translation initiator Met by methionine aminopeptidase (MetAP) is often crucial for the function and stability of proteins. On the basis of crystal structure and sequence alignment of MetAPs, we have engineered Escherichia coli MetAP by the mutation of three residues, Y168G, M206T, Q233G, in the substrate-binding pocket. Our engineered MetAPs are able to remove the Met from bulky or acidic penultimate residues, such as Met, His, Asp, Asn, Glu, Gln, Leu, Ile, Tyr, and Trp, as well as from small residues. The penultimate residue, the second residue after Met, was further removed if the antepenultimate residue, the third residue after Met, was small. By the coexpression of engineered MetAP in E. coli through the same or a separate vector, we have successfully produced recombinant proteins possessing an innate N terminus, such as onconase, an antitumor ribonuclease from the frog Rana pipiens. The N-terminal pyroglutamate of recombinant onconase is critical for its structural integrity, catalytic activity, and cyto-toxicity. On the basis of N-terminal sequence information in the protein database, 85%–90% of recombinant proteins should be produced in authentic form by our engineered MetAPs. PMID:15215523

  15. 21 CFR 582.5475 - Methionine.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Methionine. 582.5475 Section 582.5475 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary...

  16. 21 CFR 582.5475 - Methionine.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Methionine. 582.5475 Section 582.5475 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary...

  17. 21 CFR 582.5475 - Methionine.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Methionine. 582.5475 Section 582.5475 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary...

  18. 21 CFR 582.5475 - Methionine.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Methionine. 582.5475 Section 582.5475 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary...

  19. 21 CFR 582.5475 - Methionine.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Methionine. 582.5475 Section 582.5475 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary...

  20. Chemoenzymatic Synthesis of (36)S Isotopologues of Methionine and S-Adenosyl-L-methionine.

    PubMed

    Poulin, Myles B; Du, Quan; Schramm, Vern L

    2015-05-15

    Substrates containing isotope labels at specific atoms are required for transition-state analysis based on the measurement of multiple kinetic isotope effects.(36)S-labeled l-methionine and S-adenosyl-l-methionine were synthesized from elemental sulfur using a chemoenzymatic approach with >98% (36)S enrichment. This method provides access to previously inaccessible sulfur isotope-labeled substrates for sulfur kinetic isotope effect studies.

  1. Standardized ileal digestibility of proteins and amino acids in sesame expeller and soya bean meal in weaning piglets.

    PubMed

    Aguilera, A; Reis de Souza, T C; Mariscal-Landín, G; Escobar, K; Montaño, S; Bernal, M G

    2015-08-01

    Apparent ileal digestibility (AID) of diets containing sesame expeller (SE) and soya bean meal (SBM) was determined using 15 piglets (Genetiporc(®)), weaned at 17 ± 0.4 days with average body weight of 6.4 ± 0.7 kg (Fertilis 20 × G Performance, Genetiporc(®), PIC México, Querétaro, México). Piglets were randomly assigned to three treatments: (i) a reference diet with casein as the sole protein source; (ii) a mixed diet of casein-SE; and (iii) a mixed diet of casein-SBM. The chemical composition of SE and SBM was determined, and AID and standardized ileal digestibility (SID) of crude protein (CP) and amino acids (AAs) were determined for each protein source. SE contained greater quantities of ether extract, neutral detergent fibre, phytic acid, methionine and arginine than SBM. Lysine and proline contents and trypsin inhibitor activity were higher in SBM than in SE. The AID and SID of CP and AA (except for lysine and proline) were similar in SE and SBM. The AID of lysine and proline was higher in SBM than in SE (p < 0.05), and the SID of proline was higher in SE than in SBM (p < 0.05). These findings indicate that SE is an appropriate alternative protein source for early weaned pigs.

  2. Favored and disfavored pathways of protein crosslinking by glucose: glucose lysine dimer (GLUCOLD) and crossline versus glucosepane

    PubMed Central

    Nemet, Ina; Strauch, Christopher M.

    2010-01-01

    We describe the isolation and molecular characterization of a novel glucose-lysine dimer crosslink 1,3-bis-(5-amino-5-carboxypentyl)-4-(1′,2′,3′,4′-tetrahydroxybutyl)-3H-imidazolium salt, named GLUCOLD. GLUCOLD was easily formed from the Amadori product (fructose–lysine). However, when BSA was incubated with 100 mM glucose for 25 days, the levels of the lysine-lysine glucose crosslinks GLUCOLD and CROSSLINE were only 21 and <1 pmol/mg, respectively, compared to 611 pmol/mg protein for the lysine-arginine GLUCOSEPANE crosslink, in spite of more than 20 potential lysine-lysine crosslinking sites in the protein. Mechanistic investigation revealed that metal-free phosphate ions catalyzed formation of fructose–lysine and all three crosslinks from amino acids, while cationic MOPS buffer had an opposite effect. This together with the rapid formation of N6-1,4-dideoxy-5,6-dioxoglucosone derivatives by dicarbonyl trapping agents, such as 1,2-diaminobenzene or γ-guanidinobutyric acid, strongly suggests that enolization of the Amadori product and trapping of the 5,6-dioxo derivative by arginine residues constitutes the major pathway for glucose-mediated crosslinking in proteins. PMID:20607325

  3. The MurE synthetase from Thermotoga maritima is endowed with an unusual D-lysine adding activity.

    PubMed

    Boniface, Audrey; Bouhss, Ahmed; Mengin-Lecreulx, Dominique; Blanot, Didier

    2006-06-01

    The peptidoglycan of Thermotoga maritima, an extremely thermophilic eubacterium, was shown to contain no diaminopimelic acid and approximate amounts of both enantiomers of lysine (Huber, R., Langworthy, T. A., König, H., Thomm, M., Woese, C. R., Sleytr, U. B., and Stetter, K. O. (1986) Arch. Microbiol. 144, 324-333). To assess the possible involvement of the MurE activity in the incorporation of D-lysine, the murE gene from this organism was cloned in Escherichia coli, and the corresponding protein was purified as the C-terminal His6-tagged form. In vitro assays showed that D-lysine and meso-diaminopimelic acid were added to UDP-N-acetylmuramoyl-dipeptide with 25 and 10% efficiencies, respectively, relative to L-lysine. The purified enzyme was used to synthesize the L- and D-lysine-containing UDP-N-acetylmuramoyl-tripeptides; chemical analysis revealed an unusual structure for the D-lysine-containing nucleotide, namely acylation of the epsilon-amino function of D-lysine by the D-glutamyl residue. In vitro assays with MurF and MraY enzymes from T. maritima showed that this novel nucleotide was not a substrate for MurF but that it could be directly processed into tripeptide lipid I by MraY, thereby substantiating the role of MurE in the incorporation of D-lysine into peptidoglycan.

  4. Cobalamin inactivation by nitrous oxide produces severe neurological impairment in fruit bats: protection by methionine and aggravation by folates

    SciTech Connect

    van der Westhuyzen, J.; Fernandes-Costa, F.; Metz, J.

    1982-11-01

    Nitrous oxide, which inactivates cobalamin when administered to fruit bats, results in severe neurological impairment leading to ataxia, paralysis and death. This occurs after about 6 weeks in animals depleted of cobalamin by dietary restriction, and after about 10 weeks in cobalamin replete bats. Supplementation of the diet with pteroylglutamic acid caused acceleration of the neurological impairment--the first unequivocal demonstration of aggravation of the neurological lesion in cobalamin deficiency by pteroylglutamic acid. The administration of formyltetrahydropteroylglutamic acid produced similar aggravation of the neurological lesion. Supplementation of the diet with methionine protected the bats from neurological impairment, but failed to prevent death. Methionine supplementation protected against the exacerbating effect of folate, preventing the development of neurological changes. These findings lend support to the hypothesis that the neurological lesion in cobalamin deficiency may be related to a deficiency in the methyl donor S-adenosylmethionine which follows diminished synthesis of methionine.

  5. The amino acid sequence of Escherichia coli cyanase.

    PubMed

    Chin, C C; Anderson, P M; Wold, F

    1983-01-10

    The amino acid sequence of the enzyme cyanase (cyanate hydrolase) from Escherichia coli has been determined by automatic Edman degradation of the intact protein and of its component peptides. The primary peptides used in the sequencing were produced by cyanogen bromide cleavage at the methionine residues, yielding 4 peptides plus free homoserine from the NH2-terminal methionine, and by trypsin cleavage at the 7 arginine residues after acetylation of the lysines. Secondary peptides required for overlaps and COOH-terminal sequences were produced by chymotrypsin or clostripain cleavage of some of the larger peptides. The complete sequence of the cyanase subunit consists of 156 amino acid residues (Mr 16,350). Based on the observation that the cysteine-containing peptide is obtained as a disulfide-linked dimer, it is proposed that the covalent structure of cyanase is made up of two subunits linked by a disulfide bond between the single cystine residue in each subunit. The native enzyme (Mr 150,000) then appears to be a complex of four or five such subunit dimers.

  6. The Construction and Expression of Lysine-Rich Gene in the Mammary Gland of Transgenic Mice

    PubMed Central

    Ma, Xin; Zhang, Peng; Song, Guangqi; Chen, Yue; Wang, Zhongwei; Yin, Yupeng; Kong, Delong; Zhang, Sheng; Zhao, Zhihui; Ouyang, Hongsheng

    2012-01-01

    Lysine is the limiting amino acid in cereal grains, which represent a major source of human food and animal feed worldwide, and is considered the most important of the essential amino acids. In this study, β-casein, αS2-casein, and lactotransferrin cDNA clone fragments encoding lysine-rich peptides were fused together to generate a lysine-rich (LR) gene and the mammary gland-specific expression vector pBC1-LR-NEOr was constructed. Transgenic mice were generated by pronuclear microinjection of the linearized expression vectors harboring the LR transgene. The transgenic mice and their offspring were examined using multiplex polymerase chain reaction (PCR), Southern blotting, reverse transcriptase–PCR, in situ hybridization, and Western blotting techniques. Our results showed that the LR gene was successfully integrated into the mouse genome and was transmitted stably. The specific LR gene expression was restricted to the mammary gland, active alveoli of the transgenic female mice during lactation. The lysine level of the two transgenic lines was significantly higher than that of nontransgenic controls (p<0.05). In addition, the growth performance of transgenic pups was enhanced by directly feeding them the LR protein-enriched transgenic milk. Our results demonstrated that lysine-rich gene was successfully constructed and expressed in mammary gland of transgenic mice. This study will provide a better understanding of how mammary gland expression systems that increase the lysine content of milk can be applied to other mammals, such as cows. PMID:22577831

  7. Na/sup +/-dependent transport of /sup 14/C-L-lysine across bullfrog alveolar epithelium

    SciTech Connect

    Kim, K.J.; Crandall, E.D.

    1986-03-01

    Transepithelial transport of the basic amino acid L-lysine has been studied utilizing the isolated intact bullfrog lung mounted in the Ussing chamber. Lungs were excised from doubly pithed bullfrogs and sandwiched between two hemichambers. /sup 14/C-(U)-L-lysine was added to the upstream reservoir of amphibian Ringer solution, while the tissue was short-circuited. Two lungs from the same animal were used simultaneously to determine the two opposite unidirectional fluxes. Downstream and upstream radioactivities were assayed and used to estimate the apparent permeability (P) of the labeled lysine. Results indicate that the apparent P of /sup 14/C-L-lysine measured in the alveolar (M) to the pleural (S) direction is 19.06 (+- 2.84) x 10/sup -7/ cm/s and P in the S to M direction is 3.29 (+- 0.02) x 10/sup -7/ cm/s. When the 100 mM NaCl in the bath was replaced by 110 mM choline chloride, the flux of /sup 14/C-L-lysine from the alveolar to the pleural side decreased to the same value as that in the opposite direction. The flux from the pleural to the alveolar direction in the absence of Na/sup +/ did not change. These results suggest that the alveolar epithelium exhibits Na/sup +/-dependent amino acid (L-lysine) transport in the M->S, but not in the S->M, direction.

  8. Modulatory effect of curcumin on methionine-induced hyperlipidemia and hyperhomocysteinemia in albino rats.

    PubMed

    Kapoor, Puneet; Ansari, M Nazam; Bhandari, Uma

    2008-07-01

    The present study was designed to investigate the antioxidant effect of curcumin on methionine-induced hyperlipidemia and hyperhomocysteinemia in Wistar rats (200-250 g) of either sex. The vehicle control rats were treated with 1% Tween 80 in normal saline (2 ml/kg, po) for 30 days. Hyperlipidemia and hyperhomocysteinemia was induced by methionine administration (1 g/kg, po) for 30 days. A significant increase in total cholesterol, triglycerides, low density lipoprotein cholesterol (LDL-C) and homocysteine levels in serum and thiobarbituric acid reactive substances (TBARS) levels in heart homogenates were observed with a concomitant decrease in serum high density lipoprotein (HDL-C) levels in pathogenic control (i.e. group II) rats, as compared to vehicle control (i.e. group I) rats. Further, curcumin (200 mg/kg, p.o.) treatment in methionine treated rats for 30 days significantly decreased the total cholesterol, triglycerides, LDL-C and homocysteine levels in serum and TBARS levels in heart homogenates and increased serum HDL-C levels, as compared to pathogenic control (i.e. group II) rats. The results of biochemical observations were supplemented by histopathological examination of rat's aortic section. The results of test drug were comparable to that obtained with folic acid (100 mg/kg, p.o.). The results suggest that curcumin has significant antihyperlipidemic and antihyperhomocysteinemic effect against methionine-induced hyperlipidemia and hyperhomocysteinemia in rats.

  9. Protective effect of methionine supplementation on arsenic-induced alteration of glucose homeostasis.

    PubMed

    Pal, Sudipta; Chatterjee, Ajay K

    2004-05-01

    Short term exposure of arsenic produces carbohydrate depletion and hypoglycemia. Dietary deficiency of methionine causes impaired biotransformation of arsenic which has been attributed to the pathogenesis of different diseases induced by arsenic. Accordingly, the effects of methionine supplementation on the altered glucose homeostasis induced by arsenic were studied. Arsenic (as sodium arsenite) treatment (i.p) of male Wistar rats (weighing 80-100 g) at a dose of 5.55 mg kg(-1) body weight (equivalent to 35% LD50) per day for a period of 21 days caused a significant diminution in blood glucose level and fall in liver glycogen and pyruvic acid contents. The free amino acid nitrogen content of liver was elevated while that of kidney was decreased after arsenic treatment. Transaminase activities in liver and kidney were not significantly altered except that glutamate-pyruvate transaminase activity of kidney decreased significantly after arsenic treatment. Methionine supplementation reversed the above changes except decreased liver glycogen due to arsenic treatment. It may be suggested that hypoglycemia with associated decreased glycolytic activity induced by arsenic treatment at the present dose and duration can be partially counteracted by dietary methionine supplementation.

  10. Engineering a Lysine-ON Riboswitch for Metabolic Control of Lysine Production in Corynebacterium glutamicum.

    PubMed

    Zhou, Li-Bang; Zeng, An-Ping

    2015-12-18

    Riboswitches are natural RNA elements that regulate gene expression by binding a ligand. Here, we demonstrate the possibility of altering a natural lysine-OFF riboswitch from Eschericia coli (ECRS) to a synthetic lysine-ON riboswitch and using it for metabolic control. To this end, a lysine-ON riboswitch library was constructed using tetA-based dual genetic selection. After screening the library, the functionality of the selected lysine-ON riboswitches was examined using a report gene, lacZ. Selected lysine-ON riboswitches were introduced into the lysE gene (encoding a lysine transport protein) of Corynebacterium glutamicum and used to achieve dynamic control of lysine transport in a recombinant lysine-producing strain, C. glutamicum LPECRS, which bears a deregulated aspartokinase and a lysine-OFF riboswitch for dynamic control of the enzyme citrate synthase. Batch fermentation results of the strains showed that the C. glutamicum LPECRS strain with an additional lysine-ON riboswitch for the control of lysE achieved a 21% increase in the yield of lysine compared to that of the C. glutamicum LPECRS strain and even a 89% increase in yield compared to that of the strain with deregulated aspartokinase. This work provides a useful approach to generate lysine-ON riboswitches for C. glutamicum metabolic engineering and demonstrates for the first time a synergetic effect of lysine-ON and -OFF riboswitches for improving lysine production in this industrially important microorganism. The approach can be used to dynamically control other genes and can be applied to other microorganisms. PMID:26300047

  11. Design and Characterization of Auxotrophy-Based Amino Acid Biosensors

    PubMed Central

    Bertels, Felix; Merker, Holger; Kost, Christian

    2012-01-01

    Efficient and inexpensive methods are required for the high-throughput quantification of amino acids in physiological fluids or microbial cell cultures. Here we develop an array of Escherichia coli biosensors to sensitively quantify eleven different amino acids. By using online databases, genes involved in amino acid biosynthesis were identified that – upon deletion – should render the corresponding mutant auxotrophic for one particular amino acid. This rational design strategy suggested genes involved in the biosynthesis of arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, threonine, tryptophan, and tyrosine as potential genetic targets. A detailed phenotypic characterization of the corresponding single-gene deletion mutants indeed confirmed that these strains could neither grow on a minimal medium lacking amino acids nor transform any other proteinogenic amino acid into the focal one. Site-specific integration of the egfp gene into the chromosome of each biosensor decreased the detection limit of the GFP-labeled cells by 30% relative to turbidometric measurements. Finally, using the biosensors to determine the amino acid concentration in the supernatants of two amino acid overproducing E. coli strains (i.e. ΔhisL and ΔtdcC) both turbidometrically and via GFP fluorescence emission and comparing the results to conventional HPLC measurements confirmed the utility of the developed biosensor system. Taken together, our study provides not only a genotypically and phenotypically well-characterized set of publicly available amino acid biosensors, but also demonstrates the feasibility of the rational design strategy used. PMID:22829942

  12. A Methionine Deficient Diet Enhances Adipose Tissue Lipid Metabolism and Alters Anti-Oxidant Pathways in Young Growing Pigs

    PubMed Central

    Castellano, Rosa; Perruchot, Marie-Hélène; Conde-Aguilera, José Alberto; van Milgen, Jaap; Collin, Anne; Tesseraud, Sophie; Mercier, Yves; Gondret, Florence

    2015-01-01

    Methionine is a rate-limiting amino-acid for protein synthesis but non-proteinogenic roles on lipid metabolism and oxidative stress have been demonstrated. Contrary to rodents where a dietary methionine deficiency led to a lower adiposity, an increased lipid accretion rate has been reported in growing pigs fed a methionine deficient diet. This study aimed to clarify the effects of a dietary methionine deficiency on different aspects of tissue lipid metabolism and anti-oxidant pathways in young pigs. Post-weaned pigs (9.8 kg initial body weight) were restrictively-fed diets providing either an adequate (CTRL) or a deficient methionine supply (MD) during 10 days (n=6 per group). At the end of the feeding trial, pigs fed the MD diet had higher lipid content in subcutaneous adipose tissue. Expression levels of genes involved in glucose uptake, lipogenesis but also lipolysis, and activities of NADPH enzyme suppliers were generally higher in subcutaneous and perirenal adipose tissues of MD pigs, suggesting an increased lipid turnover in those pigs. Activities of the anti-oxidant enzymes superoxide dismutase, catalase and glutathione reductase were increased in adipose tissues and muscle of MD pigs. Expression level and activity of the glutathione peroxidase were also higher in liver of MD pigs, but hepatic contents in the reduced and oxidized forms of glutathione and glutathione reductase activity were lower compared with control pigs. In plasma, superoxide dismutase activity was higher but total anti-oxidant power was lower in MD pigs. These results show that a dietary methionine deficiency resulted in increased levels of lipogenesis and lipolytic indicators in porcine adipose tissues. Decreased glutathione content in the liver and coordinated increase of enzymatic antioxidant activities in adipose tissues altered the cellular redox status of young pigs fed a methionine-deficient diet. These findings illustrate that a rapidly growing animal differently adapts tissue

  13. A Methionine Deficient Diet Enhances Adipose Tissue Lipid Metabolism and Alters Anti-Oxidant Pathways in Young Growing Pigs.

    PubMed

    Castellano, Rosa; Perruchot, Marie-Hélène; Conde-Aguilera, José Alberto; van Milgen, Jaap; Collin, Anne; Tesseraud, Sophie; Mercier, Yves; Gondret, Florence

    2015-01-01

    Methionine is a rate-limiting amino-acid for protein synthesis but non-proteinogenic roles on lipid metabolism and oxidative stress have been demonstrated. Contrary to rodents where a dietary methionine deficiency led to a lower adiposity, an increased lipid accretion rate has been reported in growing pigs fed a methionine deficient diet. This study aimed to clarify the effects of a dietary methionine deficiency on different aspects of tissue lipid metabolism and anti-oxidant pathways in young pigs. Post-weaned pigs (9.8 kg initial body weight) were restrictively-fed diets providing either an adequate (CTRL) or a deficient methionine supply (MD) during 10 days (n=6 per group). At the end of the feeding trial, pigs fed the MD diet had higher lipid content in subcutaneous adipose tissue. Expression levels of genes involved in glucose uptake, lipogenesis but also lipolysis, and activities of NADPH enzyme suppliers were generally higher in subcutaneous and perirenal adipose tissues of MD pigs, suggesting an increased lipid turnover in those pigs. Activities of the anti-oxidant enzymes superoxide dismutase, catalase and glutathione reductase were increased in adipose tissues and muscle of MD pigs. Expression level and activity of the glutathione peroxidase were also higher in liver of MD pigs, but hepatic contents in the reduced and oxidized forms of glutathione and glutathione reductase activity were lower compared with control pigs. In plasma, superoxide dismutase activity was higher but total anti-oxidant power was lower in MD pigs. These results show that a dietary methionine deficiency resulted in increased levels of lipogenesis and lipolytic indicators in porcine adipose tissues. Decreased glutathione content in the liver and coordinated increase of enzymatic antioxidant activities in adipose tissues altered the cellular redox status of young pigs fed a methionine-deficient diet. These findings illustrate that a rapidly growing animal differently adapts tissue

  14. Plasma amino acid response to single test meals in humans. V. Ethiopian preschool children given lowcost protein supplements.

    PubMed

    Ljungqvist, B G; Björnesjö, K B; Gebre-Medhin, M; Habte, D; Meeuwisse, G W; Mellander, O; Svanberg, U S

    1979-02-01

    The plasma amino acid response to single test meals was studied in preschool children, aged 9 months--5 years. The amount of protein given in each test meal was 1 g per kg body weight, which represented one-third of the daily intake of the children who were recovering from protein-energy malnutrition. The test meals given was gruels made from wheat mixed with a supplementary weaning food (Faffa or Superamin) or fish protein concentrate (FPC). The plasma amino acid responses were evaluated both as PAA ratios (a modification of the Longenecker and Hause method), and as deltaMR% (percentage change in the postprandial essential amino acid molar ratios according to Graham and Placko). Both evaluation models indicated that lysine, threonine and methionine were the limiting amino acids in the Faffa/wheat diet, and that lysine and threonine were limiting in the Superamin/wheat diet. All essential amino acids seemed to be supplied in adequate amounts in the FPC/wheat diet. These results were in close agreement with the amino acid score of the diets (based on chemical analysis). PMID:424653

  15. Rat bioassays for methionine availability in 16 food sources.

    PubMed

    McDonough, F E; Bodwell, C E; Staples, R S; Wells, P A

    1989-01-01

    Methionine availabilities of 16 test proteins were assessed by comparing ten day rat growth response to the test diets and reference (casein) diets. In a preliminary study, various concentrations of methionine and cystine were fed to determine methionine requirements and effect of excess cystine. Results indicated a methionine requirement of about 550 mg per 100 g diet. Cystine had a sparing affect of 50-55%, i.e., about 300 mg could be used to meet methionine requirements. Further additions of cystine (up to 2.6 times methionine) did not affect rat growth. Methionine availabilities were excellent (88-100%) for 15 of the 16 test foods; only pinto beans (58%) were low, but prior evidence indicates that the poor growth response was due to some factor other than availability. PMID:2710754

  16. Increased methionine sulfoxide content of apoA-I in type 1 diabetes.

    PubMed

    Brock, Jonathan W C; Jenkins, Alicia J; Lyons, Timothy J; Klein, Richard L; Yim, Eunsil; Lopes-Virella, Maria; Carter, Rickey E; Thorpe, Suzanne R; Baynes, John W

    2008-04-01

    Cardiovascular disease is a major cause of morbidity and premature mortality in diabetes. HDL plays an important role in limiting vascular damage by removing cholesterol and cholesteryl ester hydroperoxides from oxidized low density lipoprotein and foam cells. Methionine (Met) residues in apolipoprotein A-I (apoA-I), the major apolipoprotein of HDL, reduce peroxides in HDL lipids, forming methionine sulfoxide [Met(O)]. We examined the extent and sites of Met(O) formation in apoA-I of HDL isolated from plasma of healthy control and type 1 diabetic subjects to assess apoA-I exposure to lipid peroxides and the status of oxidative stress in the vascular compartment in diabetes. Three tryptic peptides of apoA-I contain Met residues: Q(84)-M(86)-K(88), W(108)-M(112)-R(116), and L(144)-M(148)-R(149). These peptides and their Met(O) analogs were identified and quantified by mass spectrometry. Relative to controls, Met(O) formation was significantly increased at all three locations (Met(86), Met(112), and Met(148)) in diabetic patients. The increase in Met(O) in the diabetic group did not correlate with other biomarkers of oxidative stress, such as N(epsilon)-malondialdehyde-lysine or N(epsilon)-(carboxymethyl)lysine, in plasma or lipoproteins. The higher Met(O) content in apoA-I from diabetic patients is consistent with increased levels of lipid peroxidation products in plasma in diabetes. Using the methods developed here, future studies can address the relationship between Met(O) in apoA-I and the risk, development, or progression of the vascular complications of diabetes.

  17. Dietary crude protein has minimal effect on the activity of selected enzymes of methionine catabolism in kittens fed diets near-limiting in methionine.

    PubMed

    Strieker, M J; Morris, J G; Avery, E H; Freedland, R A; Rogers, Q R

    2008-04-01

    Previous experiments have shown that increasing the dietary crude protein (CP) of cats does not increase urea cycle enzymes or alanine amino transferase as occurs in rats. Also when an essential amino acid (EAA) is limiting in a diet for growing kittens, the kittens do not exhibit an amino acid imbalance when other EAAs are added to the diet. To study the metabolic basis for these observations which are different from that found in omnivores and herbivores, the hypothesis that increased dietary CP decreases methionine catabolism, so more is spared for growth, was tested. Fifteen male kittens were randomly assigned to one of three dietary treatments. Each diet contained 2.5 g l-methionine/kg diet and 200, 300 or 500 g CP/kg diet. The livers and kidneys were removed and assayed for methionine transaminase (MTA), cystathionase (CASE) and cystathionine synthase (CS). Free amino acid concentrations were determined in liver, kidney and plasma. The 300 and 500 g CP/kg groups had significantly greater kidney weights and body weight gains than the 200 g CP/kg group. Hepatic MTA activity was lower in the 300 than the 200 or 500 g CP/kg groups (p < 0.05). Renal MTA and CASE activities were 35% and 50% greater, respectively, for the 500 g CP/kg group than for the 200 g CP/kg diet group (p < 0.05). Renal CS activities for the 300 and 500 g CP/kg groups were 29% (p > 0.05) and 38% (p < 0.05) greater, respectively, than the 200 g CP/kg group. Cyst(e)ine concentrations were lower in the livers of the 500 g CP/kg group than the 200 g CP/kg group (p < 0.05). Cystathionine was lower in plasma and kidney from the 500 g CP/kg diet group than from the 200 g CP/kg diet group (p < 0.05). It was concluded that the metabolic basis for the increased growth of kittens fed diets marginally limiting in methionine, with increasing concentrations of dietary CP, was not mediated through decreased enzyme activity associated with the catabolism of methionine, but was the result of an increase in

  18. Effect of amino acids on the formation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in creatinine/phenylalanine and creatinine/phenylalanine/4-oxo-2-nonenal reaction mixtures.

    PubMed

    Zamora, Rosario; Alcón, Esmeralda; Hidalgo, Francisco J

    2013-12-15

    2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) formation in mixtures of creatinine, phenylalanine, amino acids and 4-oxo-2-nonenal was studied, to analyse the role of amino acids on the generation of this heterocyclic aromatic amine. When oxidised lipid was absent, cysteine, serine, aspartic acid, threonine, asparagine, tryptophan, tyrosine, proline, and methionine increased significantly (p < 0.05) the amount of PhIP formed in comparison to the control. When lipid was present, only the addition of methionine, glycine, and serine increased significantly (p < 0.05) the amount of PhIP produced, while histidine, cysteine, lysine, tryptophan, tyrosine, and alanine reduced significantly (p < 0.05) PhIP. These results may be a consequence of the different competitive reactions that occur. Thus, in the absence of lipids, thermal decomposition of the amino acids produced reactive carbonyls that converted phenylalanine into phenylacetaldehyde as a key step in the formation of PhIP. When oxidised lipid was present, amino acids competed with phenylalanine for the lipid, and amino acid degradation products were formed, among which alpha-keto acids seemed to play a role in these reactions. These results suggest that PhIP can be produced by several alternative reaction pathways from all major food components, including amino acids and lipids, in addition to carbohydrates. PMID:23993611

  19. Effect of amino acids on the formation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in creatinine/phenylalanine and creatinine/phenylalanine/4-oxo-2-nonenal reaction mixtures.

    PubMed

    Zamora, Rosario; Alcón, Esmeralda; Hidalgo, Francisco J

    2013-12-15

    2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) formation in mixtures of creatinine, phenylalanine, amino acids and 4-oxo-2-nonenal was studied, to analyse the role of amino acids on the generation of this heterocyclic aromatic amine. When oxidised lipid was absent, cysteine, serine, aspartic acid, threonine, asparagine, tryptophan, tyrosine, proline, and methionine increased significantly (p < 0.05) the amount of PhIP formed in comparison to the control. When lipid was present, only the addition of methionine, glycine, and serine increased significantly (p < 0.05) the amount of PhIP produced, while histidine, cysteine, lysine, tryptophan, tyrosine, and alanine reduced significantly (p < 0.05) PhIP. These results may be a consequence of the different competitive reactions that occur. Thus, in the absence of lipids, thermal decomposition of the amino acids produced reactive carbonyls that converted phenylalanine into phenylacetaldehyde as a key step in the formation of PhIP. When oxidised lipid was present, amino acids competed with phenylalanine for the lipid, and amino acid degradation products were formed, among which alpha-keto acids seemed to play a role in these reactions. These results suggest that PhIP can be produced by several alternative reaction pathways from all major food components, including amino acids and lipids, in addition to carbohydrates.

  20. Ontogenetic changes in digestive enzyme activities and the amino acid profile of starry flounder Platichthys stellatus

    NASA Astrophysics Data System (ADS)

    Song, Zhidong; Wang, Jiying; Qiao, Hongjin; Li, Peiyu; Zhang, Limin; Xia, Bin

    2016-09-01

    Ontogenetic changes in digestive enzyme activities and the amino acid (AA) profile of starry flounder, Platichthys stellatus, were investigated and limiting amino acids were estimated compared with the essential AA profile between larvae and live food to clarify starry flounder larval nutritional requirements. Larvae were collected at the egg stage and 0, 2, 4, 7, 12, 17, 24 days after hatching (DAH) for analysis. Larvae grew from 1.91 mm at hatching to 12.13 mm at 24 DAH. Trypsin and chymotrypsin activities changed slightly by 4 DAH and then increased significantly 4 DAH. Pepsin activity increased sharply beginning 17 DAH. Lipase activity increased significantly 4 DAH and increased progressively with larval growth. Amylase activity was also detected in newly hatched larvae and increased 7 DAH followed by a gradual decrease. High free amino acid (FAA) content was detected in starry flounder eggs (110.72 mg/g dry weight). Total FAA content dropped to 43.29 mg/g in 4-DAH larvae and then decreased gradually to 13.74 mg/g in 24-DAH larvae. Most FAAs (except lysine and methionine) decreased >50% in 4-DAH larvae compared with those in eggs and then decreased to the lowest values in 24-DAH larvae. Changes in the protein amino acid (PAA) profile were much milder than those observed for FAAs. Most PAAs increased gradually during larval development, except lysine and phenylalanine. The percentages of free threonine, valine, isoleucine, and leucine decreased until the end of the trial, whereas the protein forms of these four AAs followed the opposite trend. A comparison of the essential AA composition of live food (rotifers, Artemia nauplii, and Artemia metanauplii) and larvae suggested that methionine was potentially the first limiting AA. These results may help develop starry flounder larviculture methods by solving the AA imbalance in live food. Moreover, the increased digestive enzyme activities indicate the possibility of introducing artificial compound feed.

  1. Methionine as a potential precursor for halogenated compounds by the reaction with iron minerals

    NASA Astrophysics Data System (ADS)

    Tubbesing, C.; Krause, T.; Mulder, I.; Kotte, K.; Schöler, H. F.

    2012-04-01

    Volatile halogenated compounds (VOX) play an important role in different photochemical reactions within the troposphere and the stratosphere. Soils and sediments seem to act as a major natural source for VOX, but investigations of the reaction mechanisms are rather scarce. To get further information on potential intermediates the reaction of the amino acid methionine with the ferrous and ferric iron minerals pyrite and ferrihydrite as well as solute ferrous sulfate was studied using a gas chromatography-flame ionization detector (GC-FID). Methionine is an important amino acid in the biosynthesis of plants used as a starting compound for the messenger ethene with aminocyclopropane carboxylic acid as an intermediate product. This pathway may also occur under abiotic conditions. Ethene is assumed as precursor for various halogenated C2-compounds like vinyl chloride and dichloroethene. Due to its ubiquity by an average concentration of 10 to 290 ng/g soil and its potential to regenerate in soils and organic litter by microorganisms, methionine may be an important educt for both abiotic and biotic terrestrial halogenation processes. In laboratory tests methionine was exposed to different iron species like pyrite, iron sulfate or ferrihydrite. The oxidant H2O2 was used to start the reaction. Production values of methyl chloride and other halogenated compounds are discussed in the context of methionine as their potential precursor and several Fe-minerals as soil-borne catalysers. Several possible intermediates for the production of VOX have been detected e.g. methane, ethene or propane. A formation of isobutylene is noteworthy for some cases. In addition to VOC the production of methyl chloride and dimethyl sulfide (DMS) was observed. Only the DMS bears upon a specific mineral. The samples containing pyrite reveal the highest concentrations. To get a better assessment of methionine, respectively VOC released from methionine as precursors for halogenated compounds

  2. Dysregulated Hepatic Methionine Metabolism Drives Homocysteine Elevation in Diet-Induced Nonalcoholic Fatty Liver Disease.

    PubMed

    Pacana, Tommy; Cazanave, Sophie; Verdianelli, Aurora; Patel, Vaishali; Min, Hae-Ki; Mirshahi, Faridoddin; Quinlivan, Eoin; Sanyal, Arun J

    2015-01-01

    methyltransferase Dnmt3a decreased, the global DNA methylation was unaltered. Among individual genes, only HMG-CoA reductase (Hmgcr) was hypermethylated, and no methylation changes were observed in fatty acid synthase (Fasn), nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (Nfκb1), c-Jun, B-cell lymphoma 2 (Bcl-2) and Caspase 3. NAFLD was associated with hepatic methionine deficiency and homocysteine elevation, resulting mainly from impaired homocysteine remethylation, and aberrancy in methyltransferase reactions. Despite increased PRMT1 expression, hepatic ADMA was depleted while circulating ADMA was increased, suggesting increased export to circulation.

  3. Reconstruction of Cysteine Biosynthesis Using Engineered Cysteine-Free and Methionine-Free Enzymes

    NASA Technical Reports Server (NTRS)

    Wang, Kendrick; Fujishima, Kosuke; Abe, Nozomi; Nakahigashi, Kenji; Endy, Drew; Rothschild, Lynn J.

    2016-01-01

    Ten of the proteinogenic amino acids can be generated abiotically while the remaining thirteen require biology for their synthesis. Paradoxically, the biosynthesis pathways observed in nature require enzymes that are made with the amino acids they produce. For example, Escherichia coli produces cysteine from serine via two enzymes that contain cysteine. Here, we substituted alternate amino acids for cysteine and also methionine, which is biosynthesized from cysteine, in serine acetyl transferase (CysE) and O-acetylserine sulfhydrylase (CysM). CysE function was rescued by cysteine-and-methionine-free enzymes and CysM function was rescued by cysteine-free enzymes. Structural modeling suggests that methionine stabilizes CysM and is present in the active site of CysM. Cysteine is not conserved among CysE and CysM protein orthologs, suggesting that cysteine is not functionally important for its own synthesis. Engineering biosynthetic enzymes that lack the amino acids being synthesized provides insights into the evolution of amino acid biosynthesis and pathways for bioengineering.

  4. l-Methionine inhibits growth of human pancreatic cancer cells

    PubMed Central

    Benavides, Maximo A.; Bosland, Maarten C.; da Silva, Cássio P.; Sares, Claudia T. Gomes; de Oliveira, Alana M. Cerqueira; Kemp, Rafael; dos Reis, Rodolfo B.; Martins, Vilma R.; Sampaio, Suely V.; Bland, Kirby I.; Grizzle, William E.; dos Santos, José S.

    2015-01-01

    We have previously shown that l-methionine inhibits proliferation of breast, prostate, and colon cancer cells. This study extends these findings to BXPC-3 (mutated p53) and HPAC (wild-type p53) pancreatic cancer cells and explores the reversibility of these effects. Cells were exposed to l-methionine (5 mg/ml) for 7 days or for 3 days, followed by 4 days of culture without l-methionine (recovery). Cell proliferation, apoptosis, and cell cycle effects were assessed by flow cytometry after staining for Ki-67 or annexin V/propidium iodide. Cell proliferation was reduced by 31–35% after 7 days of methionine exposure; the effect persisted in BXPC-3 and HPAC cells after 4 days of recovery. Methionine increased apoptosis by 40–75% in HPAC cells, but not in BXPC-3 cells. Continuous exposure to methionine caused accumulation of BXPC-3 cells in the S phase and HPAC cells in both the G0/G1 and S phases; however, after 4 days of recovery, these effects disappeared. In conclusion, l-methionine inhibits proliferation and interferes with the cell cycle of BXPC-3 and HPAC pancreatic cancer cells; the effects on apoptosis remarkably persisted after methionine withdrawal. Apoptosis was induced only in BXPC-3 cells. Some of the differences in the effects of methionine between cell lines may be related to disparate p53 status. These findings warrant further studies on the potential therapeutic benefit of l-methionine against pancreatic cancer. PMID:24126240

  5. L-Methionine inhibits growth of human pancreatic cancer cells.

    PubMed

    Benavides, Maximo A; Bosland, Maarten C; da Silva, Cássio P; Gomes Sares, Claudia T; de Oliveira, Alana M Cerqueira; Kemp, Rafael; dos Reis, Rodolfo B; Martins, Vilma R; Sampaio, Suely V; Bland, Kirby I; Grizzle, William E; dos Santos, José S

    2014-02-01

    We have previously shown that L-methionine inhibits proliferation of breast, prostate, and colon cancer cells. This study extends these findings to BXPC-3 (mutated p53) and HPAC (wild-type p53) pancreatic cancer cells and explores the reversibility of these effects. Cells were exposed to L-methionine (5 mg/ml) for 7 days or for 3 days, followed by 4 days of culture without L-methionine (recovery). Cell proliferation, apoptosis, and cell cycle effects were assessed by flow cytometry after staining for Ki-67 or annexin V/propidium iodide. Cell proliferation was reduced by 31-35% after 7 days of methionine exposure; the effect persisted in BXPC-3 and HPAC cells after 4 days of recovery. Methionine increased apoptosis by 40-75% in HPAC cells, but not in BXPC-3 cells. Continuous exposure to methionine caused accumulation of BXPC-3 cells in the S phase and HPAC cells in both the G0/G1 and S phases; however, after 4 days of recovery, these effects disappeared. In conclusion, L-methionine inhibits proliferation and interferes with the cell cycle of BXPC-3 and HPAC pancreatic cancer cells; the effects on apoptosis remarkably persisted after methionine withdrawal. Apoptosis was induced only in BXPC-3 cells. Some of the differences in the effects of methionine between cell lines may be related to disparate p53 status. These findings warrant further studies on the potential therapeutic benefit of L-methionine against pancreatic cancer.

  6. Lysine fortification of wheat flour improves selected indices of the nutritional status of predominantly cereal-eating families in Pakistan.

    PubMed

    Hussain, Tajammal; Abbas, Shaid; Khan, Mushtaq A; Scrimshaw, Nevin S

    2004-06-01

    Wheat provides more than 50% of the protein and calorie intake of the population of Pakistan. Legumes and animal protein that could complement the amino acid pattern of wheat, in which lysine is the first limiting amino acid for utilization of protein, are not affordable by members of lower socioeconomic groups in developing countries. The purpose of the study was to determine whether lysine fortification of wheat flour would have a positive impact on populations consuming a predominantly wheat-based diet. A double-blind study was carried out for three months on the outskirts of Peshawar, Pakistan. Forty families received wheat flour fortified with lysine, and 40 families received wheat flour without lysine. Wheat provided 59% of the protein for men, 65% for women, and 58% for children. The weight and height of the children in both groups increased during the study, but the increase was significantly greater in the lysine group. Hemoglobin increased significantly in the women receiving lysine-fortified flour. Transferrin levels increased significantly in men, women, and children in the lysine group as compared with those in the control group. Prealbumin increased significantly in adults receiving additional lysine but decreased in children. Men, women, and children in the lysine-supplemented families had significant increases in CD4, CD8, and complement C3 as compared with controls. These results indicate that lysine fortification of wheat flour can significantly improve sensitive indicators of nutritional status in a population consuming a diet in which 58% to 65% of the protein, depending on age and sex, is supplied by wheat.

  7. Redox regulation of methionine aminopeptidase 2 activity.

    PubMed

    Chiu, Joyce; Wong, Jason W H; Hogg, Philip J

    2014-05-23

    Protein translation is initiated with methionine in eukaryotes, and the majority of proteins have their N-terminal methionine removed by methionine aminopeptidases (MetAP1 and MetAP2) prior to action. Methionine removal can be important for protein function, localization, or stability. No mechanism of regulation of MetAP activity has been identified. MetAP2, but not MetAP1, contains a single Cys(228)-Cys(448) disulfide bond that has an -RHStaple configuration and links two β-loop structures, which are hallmarks of allosteric disulfide bonds. From analysis of crystal structures and using mass spectrometry and activity assays, we found that the disulfide bond exists in oxidized and reduced states in the recombinant enzyme. The disulfide has a standard redox potential of -261 mV and is efficiently reduced by the protein reductant, thioredoxin, with a rate constant of 16,180 m(-1) s(-1). The MetAP2 disulfide bond also exists in oxidized and reduced states in glioblastoma tumor cells, and stressing the cells by oxygen or glucose deprivation results in more oxidized enzyme. The Cys(228)-Cys(448) disulfide is at the rim of the active site and is only three residues distant from the catalytic His(231), which suggested that cleavage of the bond would influence substrate hydrolysis. Indeed, oxidized and reduced isoforms have different catalytic efficiencies for hydrolysis of MetAP2 peptide substrates. These findings indicate that MetAP2 is post-translationally regulated by an allosteric disulfide bond, which controls substrate specificity and catalytic efficiency.

  8. Reinventing cell penetrating peptides using glycosylated methionine sulfonium ion sequences

    SciTech Connect

    Kramer, Jessica R.; Schmidt, Nathan W.; Mayle, Kristine M.; Kamei, Daniel T.; Wong, Gerard C.L.; Deming, Timothy J.

    2015-04-15

    Cell penetrating peptides (CPPs) are intriguing molecules that have received much attention, both in terms of mechanistic analysis and as transporters for intracellular therapeutic delivery. Most CPPs contain an abundance of cationic charged residues, typically arginine, where the amino acid compositions, rather than specific sequences, tend to determine their ability to enter cells. Hydrophobic residues are often added to cationic sequences to create efficient CPPs, but typically at the penalty of increased cytotoxicity. Here, we examined polypeptides containing glycosylated, cationic derivatives of methionine, where we found these hydrophilic polypeptides to be surprisingly effective as CPPs and to also possess low cytotoxicity. X-ray analysis of how these new polypeptides interact with lipid membranes revealed that the incorporation of sterically demanding hydrophilic cationic groups in polypeptides is an unprecedented new concept for design of potent CPPs.

  9. Reinventing cell penetrating peptides using glycosylated methionine sulfonium ion sequences

    DOE PAGES

    Kramer, Jessica R.; Schmidt, Nathan W.; Mayle, Kristine M.; Kamei, Daniel T.; Wong, Gerard C.L.; Deming, Timothy J.

    2015-04-15

    Cell penetrating peptides (CPPs) are intriguing molecules that have received much attention, both in terms of mechanistic analysis and as transporters for intracellular therapeutic delivery. Most CPPs contain an abundance of cationic charged residues, typically arginine, where the amino acid compositions, rather than specific sequences, tend to determine their ability to enter cells. Hydrophobic residues are often added to cationic sequences to create efficient CPPs, but typically at the penalty of increased cytotoxicity. Here, we examined polypeptides containing glycosylated, cationic derivatives of methionine, where we found these hydrophilic polypeptides to be surprisingly effective as CPPs and to also possess lowmore » cytotoxicity. X-ray analysis of how these new polypeptides interact with lipid membranes revealed that the incorporation of sterically demanding hydrophilic cationic groups in polypeptides is an unprecedented new concept for design of potent CPPs.« less

  10. Proteome-wide analysis of lysine acetylation in the plant pathogen Botrytis cinerea

    PubMed Central

    Lv, Binna; Yang, Qianqian; Li, Delong; Liang, Wenxing; Song, Limin

    2016-01-01

    Lysine acetylation is a dynamic and reversible post-translational modification that plays an important role in diverse cellular processes. Botrytis cinerea is the most thoroughly studied necrotrophic species due to its broad host range and huge economic impact. However, to date, little is known about the functions of lysine acetylation in this plant pathogen. In this study, we determined the lysine acetylome of B. cinerea through the combination of affinity enrichment and high-resolution LC-MS/MS analysis. Overall, 1582 lysine acetylation sites in 954 proteins were identified. Bioinformatics analysis shows that the acetylated proteins are involved in diverse biological functions and show multiple cellular localizations. Several particular amino acids preferred near acetylation sites, including KacY, KacH, Kac***R, KacF, FKac and Kac***K, were identified in this organism. Protein interaction network analysis demonstrates that a variety of interactions are modulated by protein acetylation. Interestingly, 6 proteins involved in virulence of B. cinerea, including 3 key components of the high-osmolarity glycerol pathway, were found to be acetylated, suggesting that lysine acetylation plays regulatory roles in pathogenesis. These data provides the first comprehensive view of the acetylome of B. cinerea and serves as a rich resource for functional analysis of lysine acetylation in this plant pathogen. PMID:27381557

  11. Crystal Growth, Thermal, Optical, and Dielectric Properties of L-Lysine Doped Kdp Crystals

    NASA Astrophysics Data System (ADS)

    Parikh, Ketan D.; Dave, Dipak J.; Joshi, Mihir J.

    Single crystals of pure and various amount of L-lysine doped KDP crystals were grown from aqueous solution. The doping of L-lysine was confirmed by CHN analysis and FT-IR spectroscopy. Powder XRD was carried out to assess the single phase nature of the samples. The effect of doping on thermal stability of the crystals was carried out by TGA and the kinetic and thermodynamic parameters of dehydration were evaluated. It was found that as the amount of doping of amino acid, L-lysine, increased the thermal stability of the grown crystals decreased. However, the second-harmonic generation (SHG) efficiency of Nd:YAG laser and UV-vis spectroscopy studies indicated that as the L-lysine doping increased in KDP crystals the SHG efficiency and optical transmission percentage increased. The dielectric constant and the dielectric loss of L-lysine doped KDP crystals are lower than the pure KDP crystals. Hence L-lysine doped KDP crystals are found to be more beneficial from an application point of view as compared to pure KDP crystals. The results are discussed.

  12. Lysine acetylation stabilizes SP2 protein in the silkworm Bombyx mori.

    PubMed

    Zhou, Yong; Wu, Chengcheng; Sheng, Qing; Jiang, Caiying; Chen, Qin; Lv, Zhengbing; Yao, Juming; Nie, Zuoming

    2016-01-01

    Lysine acetylation (Kac) is a vital post-translational modification that plays an important role in many cellular processes in organisms. In the present study, the nutrient storage proteins in hemolymph were first found to be highly acetylated-particularly SP2 protein, which contains 20 potential Kac sites. Further results confirmed that lysine acetylation could stabilize and up-regulate the protein level of anti-apoptosis protein SP2, thereby improving the survival of H2O2-treated BmN cells and suppressing the apoptosis induced by H2O2. The potential mechanism involved in the inhibition of ubiquitin-mediated proteasomal degradation by crosstalk between lysine acetylation and ubiquitination. Our results showed that the increase in the acetylation level by TSA could decrease the ubiquitination and improve the protein level of SP2, indicating that lysine acetylation could influence the SP2 protein level through competition between ubiquitination and the suppression of ubiquitin-mediated proteasomal degradation, thereby stabilizing the protein. SP2 is a major nutrient storage protein from hemolymph for amino acid storage and utilization. The crosstalk between lysine acetylation and ubiquitination of SP2 might imply an important role of lysine acetylation for nutrient storage and utilization in silkworm. PMID:27374983

  13. Molecular genetics of hepatic methionine adenosyltransferase deficiency.

    PubMed

    Chou, J Y

    2000-01-01

    Hepatic methionine adenosyltransferase (MAT) deficiency is caused by mutations in the human MAT1A gene that abolish or reduce hepatic MAT activity that catalyzes the synthesis of S-adenosylmethionine from methionine and ATP. This genetic disorder is characterized by isolated persistent hypermethioninemia in the absence of cystathionine beta-synthase deficiency, tyrosinemia, or liver disease. Depending on the nature of the genetic defect, hepatic MAT deficiency can be transmitted either as an autosomal recessive or dominant trait. Genetic analyses have revealed that mutations identified in the MAT1A gene only partially inactivate enzymatic activity, which is consistent with the fact that most hepatic MAT-deficient individuals are clinically well. Two hypermethioninemic individuals with null MAT1A mutations have developed neurological problems, including brain demyelination, although this correlation is by no means absolute. Presently, it is recommended that a DNA-based diagnosis should be performed for isolated hypermethioninemic individuals with unusually high plasma methionine levels to assess if therapy aimed at the prevention of neurological manifestations is warranted.

  14. Methionine restriction beyond life-span extension.

    PubMed

    Ables, Gene P; Hens, Julie R; Nichenametla, Sailendra N

    2016-01-01

    Dietary methionine restriction (MR) extends life span across species via various intracellular regulatory mechanisms. In rodents, MR induces resistance against adiposity, improves hepatic glucose metabolism, preserves cardiac function, and reduces body size, all of which can affect the onset of age-related diseases. Recent studies have shown that MR-affected biomarkers, such as fibroblast growth factor 21, adiponectin, leptin, cystathionine β synthase, and insulin-like growth factor 1, can potentially alter physiology. The beneficial effects of MR could be explained in part by its ability to reduce mitochondrial oxidative stress. Studies have revealed that MR can reduce reactive oxygen species that damage cells and promote cancer progression. It has been demonstrated that either MR or the targeting of specific genes in the methionine cycle could induce cell apoptosis while decreasing proliferation in several cancer models. The complete mechanism underlying the actions of MR on the cell cycle during cancer has not been fully elucidated. Epigenetic mechanisms, such as methylation and noncoding RNAs, are also possible downstream effectors of MR; future studies should help to elucidate some of these mechanisms. Despite evidence that changes in dietary methionine can affect epigenetics, it remains unknown whether epigenetics is a mechanism in MR. This review summarizes research on MR and its involvement in metabolism, cancer, and epigenetics.

  15. Carbon-11-methionine and PET in evaluation of treatment response of breast cancer.

    PubMed Central

    Huovinen, R.; Leskinen-Kallio, S.; Någren, K.; Lehikoinen, P.; Ruotsalainen, U.; Teräs, M.

    1993-01-01

    Uptake of L-methyl-11C-methionine (11C-methionine) in breast cancer metastases was studied with positron emission tomography (PET). Eight patients with soft tissue metastases were studied twice: before the onset of chemotherapy (4), hormonal therapy (3) or radiotherapy (1) and 3-14 weeks later. The radioactivity concentration of the low molecular weight fraction of venous plasma samples separated by fast gel filtration was used as input function. The input corrected uptake rate of 11C-methionine (Ki) in breast cancer metastases before the treatment ranged between 0.035 and 0.186 1 min-1 and the standardised uptake value (SUV) between 2.0 and 11.4. The uptake of 11C-methionine into the metastases decreased when clinical objective stability or regression of the metastases was later obtained and increased in cases where progressive disease was seen during treatment. We conclude that metabolic changes in the amino acid metabolism detected by PET precede the clinical response, and may be of clinical value in predicting the treatment response. Images Figure 1 PMID:8471437

  16. Effect of dietary lysine restriction and arginine supplementation in two patients with pyridoxine-dependent epilepsy.

    PubMed

    Yuzyuk, Tatiana; Thomas, Amanda; Viau, Krista; Liu, Aiping; De Biase, Irene; Botto, Lorenzo D; Pasquali, Marzia; Longo, Nicola

    2016-07-01

    Pyridoxine-Dependent Epilepsy (PDE) is a recessive disorder caused by deficiency of α-aminoadipic semialdehyde dehydrogenase in the catabolic pathway of lysine. It is characterized by intractable seizures controlled by the administration of pharmacological doses of vitamin B6. Despite seizure control with pyridoxine, intellectual disability and developmental delays are still observed in some patients with PDE, likely due to the accumulation of toxic intermediates in the lysine catabolic pathway: alpha-aminoadipic semialdehyde (AASA), delta-1-piperideine-6-carboxylate (P6C), and pipecolic acid. Here we evaluate biochemical and clinical parameters in two PDE patients treated with a lysine-restricted diet and arginine supplementation (100-150mg/kg), aimed at reducing the levels of PDE biomarkers. Lysine restriction resulted in decreased accumulation of PDE biomarkers and improved development. Plasma lysine but not plasma arginine, directly correlated with plasma levels of AASA-P6C (p<0.001, r(2)=0.640) and pipecolic acid (p<0.01, r(2)=0.484). In addition, plasma threonine strongly correlated with the levels of AASA-P6C (p<0.0001, r(2)=0.732) and pipecolic acid (p<0.005, r(2)=0.527), suggesting extreme sensitivity of threonine catabolism to pyridoxine availability. Our results further support the use of dietary therapies in combination with pyridoxine for the treatment of PDE. PMID:27324284

  17. Accessibility and mobility of lysine residues in. beta. -lactoglobulin

    SciTech Connect

    Brown, E.M.; Pfeffer, P.E.; Kumosinski, T.F.; Greenberg, R.

    1988-07-26

    N/sup epsilon/-(/sup 2/H/sub 6/)Isopropyllysyl-..beta..-lactoglobulin was prepared by reductive alkylation of ..beta..-lactoglobulin with (/sup 2/H/sub 6/)acetone and NaBH/sub 4/ to provide a /sup 2/H (NMR) probe for the study of lysine involvement in lipid-protein interactions. Amino acid analysis showed 80% of the protein's 15 lysine residues to be labeled. Unmodified lysine residues were located through peptide maps produced from CNBr, tryptic, and chymotryptic digests of the labeled protein. Average correlation times calculated from /sup 2/H NMR spectra were 20 and 320 ps for 8.7 and 3.3 residues, respectively, in 6 M guanidine hydrochloride; in nondenaturing solution, values of 70 and 320 ps were obtained for 6.5 and 3.2 residues, respectively, with the remaining 2.3 modified residues not observed, suggesting that side chains of lysine residues in unordered or flexible regions were more mobile than those in stable periodic structures. /sup 2/H NMR spectra of the protein complexed with dipalmitoylphosphatidylcholine confirmed the extrinsic membrane protein type behavior of ..beta..-lactoglobulin previously reported from /sup 31/P NMR studies of the phospholipids complexed with ..beta..-lactoglobulin. Although no physiological function has yet been identified, comparison of these results with the X-ray structure supports the hypothesis that residues not accessible for modification may help to stabilize the cone-shaped ..beta..-barrel thought to contain binding sites for small lipid-soluble molecules.

  18. Effect of supplementation of crystalline lysine on the performance of WL layers in tropics during summer.

    PubMed

    Kumari, K Naga Raja; Reddy, V Ravinder; Preetham, V Chinni; Kumar, D Srinivas; Sen, Arup Ratan; Rao, S Venkata Rama

    2016-04-01

    A trial was conducted to evaluate the effect of lysine concentration in the diet of WL layers with constant ratio of other essential amino acids to lysine. Pullets (528) aged 25 to 36 weeks were fed with test diet containing two protein levels (13.36 and 15.78%) each with 5% concentration of lysine (0.50, 0.55, 0.60, 0.65, and 0.70) and a control with 17% CP and 0.70%, lysine. Each test diet was fed ad libitum to six replicates of eight birds for a period of 12 weeks. Egg production (EP), egg weight (EW), egg mass (EM), feed efficiency (g/g) (FE), body weight gain (BWG), Haugh unit (HU) and yolk colour (YC) were measured. Increased (P ≤ 0.05) EP, EW, EM, FE and BWG were obtained with increasing lysine concentration in diets. Whereas, feed intake/h/day, feed intake/egg, egg shell defects (ESD), mortality and shell thickness were not affected (P ≥ 0.05) by the concentration of lysine in diet. However, higher (P ≤ 0.05) HU score and YC were noticed at low lysine (0.50 %) concentrations. Based on this, it was concluded that WL layers (25-36 weeks) reared in open-sided houses in the tropics require approximately 0.70 % lysine (597.90 vs. 584.39 mg/h/day) in low (13.36% CP) and high (15.78% CP) protein groups in diets containing approximately 2700 kcal of ME/kg in summer.

  19. Comparison of the effects of seleno-l-methionine, seleno-dl-methionine, and selenized yeast on reproduction of mallards

    USGS Publications Warehouse

    Heinz, G.H.; Hoffman, D.J.

    1996-01-01

    The toxicities of seleno-L-methionine, seleno-DL-methionine, and selenized yeast were compared. Ten pairs of mallards were fed a control diet and 15 pairs were fed diets containing 10 ppm selenium as seleno-DL-methionine, seleno-L-methionine, or selenized yeast. Hatching of fertile eggs was significantly lower for females fed 10 ppm selenium as seleno-DL-methionine (7.6%) and seleno-L-methionine (6.4%) than for controls (41.3%). Survival of ducklings was lower when their parents had been fed 10 ppm selenium as seleno-L-methionine (20.0%) than for controls (98.4%). The number of 6-day-old ducklings produced per female was significantly lower for mallards fed 10 ppm selenium as seleno-DL-methionine (0.47) or selenized yeast (2.67) than for controls (6.10), and was significantly lower for mallards fed seleno-L-methionine (0.13) than for mallards fed selenized yeast. The eighth eggs of females fed the DL or L forms of selenomethionine contained means of 9.2 and 8.9 ppm selenium, wet weight; these means were higher than the mean (6.6 ppm) for females fed selenized yeast. Among embryos that died at 7 days of age or older, the percentage of embryos that were deformed was 1.3% for controls, 24.6% for seleno-DL-methionine, 28.2% for seleno-L-methionine, and 11.0% for selenized yeast. The results suggested that seleno-DL-methionine and seleno-L-methionine were of similar toxicity and were both more toxic than selenium from selenized yeast.

  20. SPOTing Acetyl-Lysine Dependent Interactions.

    PubMed

    Picaud, Sarah; Filippakopoulos, Panagis

    2015-08-17

    Post translational modifications have been recognized as chemical signals that create docking sites for evolutionary conserved effector modules, allowing for signal integration within large networks of interactions. Lysine acetylation in particular has attracted attention as a regulatory modification, affecting chromatin structure and linking to transcriptional activation. Advances in peptide array technologies have facilitated the study of acetyl-lysine-containing linear motifs interacting with the evolutionary conserved bromodomain module, which specifically recognizes and binds to acetylated sequences in histones and other proteins. Here we summarize recent work employing SPOT peptide technology to identify acetyl-lysine dependent interactions and document the protocols adapted in our lab, as well as our efforts to characterize such bromodomain-histone interactions. Our results highlight the versatility of SPOT methods and establish an affordable tool for rapid access to potential protein/modified-peptide interactions involving lysine acetylation.

  1. SPOTing Acetyl-Lysine Dependent Interactions

    PubMed Central

    Picaud, Sarah; Filippakopoulos, Panagis

    2015-01-01

    Post translational modifications have been recognized as chemical signals that create docking sites for evolutionary conserved effector modules, allowing for signal integration within large networks of interactions. Lysine acetylation in particular has attracted attention as a regulatory modification, affecting chromatin structure and linking to transcriptional activation. Advances in peptide array technologies have facilitated the study of acetyl-lysine-containing linear motifs interacting with the evolutionary conserved bromodomain module, which specifically recognizes and binds to acetylated sequences in histones and other proteins. Here we summarize recent work employing SPOT peptide technology to identify acetyl-lysine dependent interactions and document the protocols adapted in our lab, as well as our efforts to characterize such bromodomain-histone interactions. Our results highlight the versatility of SPOT methods and establish an affordable tool for rapid access to potential protein/modified-peptide interactions involving lysine acetylation. PMID:27600229

  2. SPOTing Acetyl-Lysine Dependent Interactions

    PubMed Central

    Picaud, Sarah; Filippakopoulos, Panagis

    2015-01-01

    Post translational modifications have been recognized as chemical signals that create docking sites for evolutionary conserved effector modules, allowing for signal integration within large networks of interactions. Lysine acetylation in particular has attracted attention as a regulatory modification, affecting chromatin structure and linking to transcriptional activation. Advances in peptide array technologies have facilitated the study of acetyl-lysine-containing linear motifs interacting with the evolutionary conserved bromodomain module, which specifically recognizes and binds to acetylated sequences in histones and other proteins. Here we summarize recent work employing SPOT peptide technology to identify acetyl-lysine dependent interactions and document the protocols adapted in our lab, as well as our efforts to characterize such bromodomain-histone interactions. Our results highlight the versatility of SPOT methods and establish an affordable tool for rapid access to potential protein/modified-peptide interactions involving lysine acetylation.

  3. Potent and sustained cellular inhibition of miR-122 by lysine-derivatized peptide nucleic acids (PNA) and phosphorothioate locked nucleic acid (LNA)/2'-O-methyl (OMe) mixmer anti-miRs in the absence of transfection agents

    PubMed Central

    Torres, Adrian G.; Threlfall, Richard N.

    2011-01-01

    Efficient cell delivery of antisense oligonucleotides (ONs) is a key issue for their potential therapeutic use. It has been shown recently that some ONs can be delivered into cells without the use of transfection agents (gymnosis), but this generally requires cell incubation over several days and high amounts of ONs (micromolar concentrations). Here we have targeted microRNA 122 (miR-122), a small non-coding RNA involved in regulation of lipid metabolism and in the replication of hepatitis C virus, with ONs of different chemistries (anti-miRs) by gymnotic delivery in cell culture. Using a sensitive dual-luciferase reporter assay, anti-miRs were screened for their ability to enter liver cells gymnotically and inhibit miR-122 activity. Efficient miR-122 inhibition was obtained with cationic PNAs and 2'-O-methyl (OMe) and Locked Nucleic Acids (LNA)/OMe mixmers containing either phosphodiester (PO) or phosphorothioate (PS) linkages at sub-micromolar concentrations when incubated with cells for just 4 hours. Furthermore, PNA and PS-containing anti-miRs were able to sustain miR-122 inhibitory effects for at least 4 days. LNA/OMe PS anti-miRs were the most potent anti-miR chemistry tested in this study, an ON chemistry that has been little exploited so far as anti-miR agents towards therapeutics. PMID:22567190

  4. Extending the cross-linking/mass spectrometry strategy: Facile incorporation of photo-activatable amino acids into the model protein calmodulin in Escherichia coli cells.

    PubMed

    Piotrowski, Christine; Ihling, Christian H; Sinz, Andrea

    2015-11-01

    Photo-induced cross-linking is a highly promising technique to investigate protein conformations and protein-protein interactions in their natural cellular environment. One strategy relies on the non-directed incorporation of diazirine-containing photo-activatable amino acids into proteins and a subsequent cross-link formation induced by UV-A irradiation. The advantage of this photo-cross-linking strategy is that it is not restricted to lysine residues and that hydrophobic regions in proteins can also be targeted, which is advantageous for investigating membrane proteins. Here, we present a simplified protocol that relies on the use of mineral salts medium without any special requirements for the incorporation of photo-methionines into proteins in Escherichia coli cells. The possibility to perform these experiments in E. coli is especially valuable as it is the major system for recombinant protein production. The method is exemplified for the Ca(2+) regulating protein calmodulin containing nine methionines, which were found to be replaced by their photo-activatable analogues. Our protocol allows the facile and stochastic incorporation of photo-methionines as the basis for conducting photo-cross-linking experiments in E. coli in an efficient manner.

  5. A Method to determine lysine acetylation stoichiometries

    SciTech Connect

    Nakayasu, Ernesto S.; Wu, Si; Sydor, Michael A.; Shukla, Anil K.; Weitz, Karl K.; Moore, Ronald J.; Hixson, Kim K.; Kim, Jong Seo; Petyuk, Vladislav A.; Monroe, Matthew E.; Pasa-Tolic, Ljiljana; Qian, Weijun; Smith, Richard D.; Adkins, Joshua N.; Ansong, Charles

    2014-07-21

    A major bottleneck to fully understanding the functional aspects of lysine acetylation is the lack of stoichiometry information. Here we describe a mass spectrometry method using a combination of isotope labeling and detection of a diagnostic fragment ion to determine the stoichiometry of lysine acetylation on proteins globally. Using this technique, we determined the modification occupancy on hundreds of acetylated peptides from cell lysates and cross-validated the measurements via immunoblotting.

  6. Dietary L-methionine restriction decreases oxidative stress in porcine liver mitochondria.

    PubMed

    Ying, Yang; Yun, Ji; Guoyao, Wu; Kaiji, Sun; Zhaolai, Dai; Zhenlong, Wu

    2015-05-01

    Dietary methionine restriction (MetR) has been reported to improve hepatocyte function in mammals. However, the underlying mechanisms remain largely unknown. This study was conducted with a swine model to test the hypothesis that MetR decreases generation of reactive oxygen species (ROS) and attenuates oxidative damage in hepatic mitochondria. Twenty-four 35-day old pigs were fed a control diet or a Met-restricted diet for two weeks. Liver mitochondria were isolated to determine: 8-oxodG in mitochondrial DNA, oxidative-derived proteins markers, including glutamic semialdehyde (GSA), aminoadipic semialdehydes (AASA), carboxyethyl-lysine (CEL), carboxymethyl-lysine (CML), and malondialdehyde lysine (MDAL), mitochondrial H2O2 generation rate; rates of oxygen consumption; free radical leak (FRL); anti-oxidative capacity, electron transport complex activity; and protein abundances of respiratory chain complex subunits (NDUFA9, SDHA, Core 2, and Cox 1), manganese superoxide dismutase (MnSOD), and apoptosis-inducing factor (AIF). Compared with the control, MetR decreased mitochondrial 8-oxodG content, H2O2 generation, FRL (P<0.05), and increased rates of oxygen consumption. Abundances of markers for protein oxidative damage, including GSA, AASA, CEL, and CML, were decreased (P<0.05) by 40%, 30%, 32%, and 28%, respectively, compared with the control. Western blot analysis revealed that MetR decreased (P<0.05) the protein abundances of complex subunits, NDUFA9 and AIF without affecting expression of SDHA, Core 2, Cox 1 or MnSOD. The complex I activity (P<0.05) were lowered in MetR group as compared with that of control. Collectively, our findings indicate that dietary MetR decreases mitochondrial ROS generation primarily via inhibiting complex I activity and ROS generation rather than augmenting anti-oxidative capacity, thereby ameliorating oxidative damage to hepatic mitochondrial DNA and proteins.

  7. Co-expression of bacterial aspartate kinase and adenylylsulfate reductase genes substantially increases sulfur amino acid levels in transgenic alfalfa (Medicago sativa L.).

    PubMed

    Tong, Zongyong; Xie, Can; Ma, Lei; Liu, Liping; Jin, Yongsheng; Dong, Jiangli; Wang, Tao

    2014-01-01

    Alfalfa (Medicago sativa L.) is one of the most important forage crops used to feed livestock, such as cattle and sheep, and the sulfur amino acid (SAA) content of alfalfa is used as an index of its nutritional value. Aspartate kinase (AK) catalyzes the phosphorylation of aspartate to Asp-phosphate, the first step in the aspartate family biosynthesis pathway, and adenylylsulfate reductase (APR) catalyzes the conversion of activated sulfate to sulfite, providing reduced sulfur for the synthesis of cysteine, methionine, and other essential metabolites and secondary compounds. To reduce the feedback inhibition of other metabolites, we cloned bacterial AK and APR genes, modified AK, and introduced them into alfalfa. Compared to the wild-type alfalfa, the content of cysteine increased by 30% and that of methionine increased substantially by 60%. In addition, a substantial increase in the abundance of essential amino acids (EAAs), such as aspartate and lysine, was found. The results also indicated a close connection between amino acid metabolism and the tricarboxylic acid (TCA) cycle. The total amino acid content and the forage biomass tested showed no significant changes in the transgenic plants. This approach provides a new method for increasing SAAs and allows for the development of new genetically modified crops with enhanced nutritional value. PMID:24520364

  8. Isolation and primary structure of a methionine- and cystine-rich seed protein of Cannabis sativa.

    PubMed

    Odani, S; Odani, S

    1998-04-01

    A 10-kDa protein was isolated from resting seeds of hemp (Cannabis sativa) by buffer extraction, gel filtration, ion-exchange chromatography, and reversed-phase high-pressure liquid chromatography. The protein did not inhibit bovine trypsin. It consisted of subunits composed of 27 and 61 residues and was held together by two disulfide bonds. The complete amino acid sequence was identified by protein analysis, and had 20 mole% of amino acids containing sulfur. The protein was most similar to a methionine-rich protein of Brazil nut (Bertholletia excelsa) and to Mabinlin IV, a sweetness-inducing protein of Capparis masaikai. The high methionine content and the absence of trypsin inhibitory activity suggested that the seed protein can be used to improve the nutritional quality of plant food-stuffs.

  9. Quantitative analysis of the interaction between l-methionine derivative and oligonucleotides.

    PubMed

    Mota, Élia; Sousa, Fani; Queiroz, João A; Cruz, Carla

    2015-04-01

    This study explores the use of l-methionine derivative as a potential affinity ligand for nucleic acids purification. The l-methionine derivative is synthesized by activation of the carboxylic acid group with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide follow by immobilization on amine sensor surface, previously activated and treated with ethylenediamine. Their affinity towards oligonucleotides has been determined by surface plasmon resonance biosensor. The highest affinity is found for cytosine and thymine, followed by adenine, whereas the lowest affinity is found for guanine. For hetero-oligonucleotides the affinity order is CCCTTT > CCCAAA ≈ AAATTT > GGGTTT, showing that nucleotides with cytosine have the highest affinity, and the presence of guanine reduces the affinity, corroborating with the results obtained with homo-oligonucleotides.

  10. One-pot modification of 5'-capped RNA based on methionine analogs.

    PubMed

    Muttach, Fabian; Rentmeister, Andrea

    2016-09-01

    This paper outlines chemically and enzymatically synthesized S-adenosylmethionine (AdoMet) analogs and their use in the site-specific modification of RNA by methyltransferases, enabling the facile attachment of clickable moieties to the nucleic acid. We then focus on methodological aspects of setting up a methyltransferase-based enzymatic cascade reaction starting from methionine analogs. This strategy is applied to the one-pot modification of the mRNA cap which is subsequently derivatized in copper-free and copper-catalyzed click reactions. We show that high transfer efficiencies to the cap are obtained using Se-propargyl-, hexenynyl- and azido-bearing methionine analogs. By switching to other methyltransferases our one-pot modification approach should be directly applicable to the regiospecific modification of other target molecules including nucleic acids, proteins and small molecules.

  11. 9 CFR 319.5 - Mechanically Separated (Species).

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... Federal Register in accordance with 5 U.S.C. 552(a) and 1 CFR Part 51. ... paragraph, essential amino acid content includes isoleucine, leucine, lysine, methionine, phenylalanine..., methionine, phenylalanine, threonine, valine, tyrosine, arginine, histidine, alanine, aspartic acid,...

  12. Amino Acid and Vitamin Requirements of Several Bacteroides Strains

    PubMed Central

    Quinto, Grace

    1966-01-01

    Nutritional studies were performed on nine Bacteroides strains, by use of the methodology and media of anaerobic rumen microbiology. Ristella perfoetens CCI required l-arginine hydrochloride, l-tryptophan, l-leucine, l-histidine hydrochloride, l-cysteine hydrochloride, dl-valine, dl-tyrosine, and the vitamin calcium-d-pantothenate, since scant turbidity developed in media without these nutrients. R. perfoetens was stimulated by glycine, dl-lysine hydrochloride, dl-isoleucine, l-proline, l-glutamic acid, dl-alanine, dl-phenylalanine, dl-methionine, and the vitamins nicotinamide and p-aminobenzoic acid, since maximal turbidity developed more slowly in media without these nutrients than in complete medium. Medium A-23, which was devised for R. perfoetens, contained salts, 0.0002% nicotinamide and calcium d-pantothenate, 0.00001% p-aminobenzoic acid, 0.044% l-tryptophan, 0.09% l-glutamic acid, and 0.1% of the other 13 amino acids listed above. Zuberella clostridiformis and seven strains of R. pseudoinsolita did not require vitamins, and showed no absolute requirement for any one amino acid. Various strains produced maximal turbidity more slowly in media deficient in l-proline, glycine, l-glutamic acid, dl-serine, l-histidine hydrochloride, dl-alanine, or l-cysteine hydrochloride, than in complete medium. These eight strains grew optimally in medium A-23 plus 0.1% dl-serine but without vitamins. PMID:16349673

  13. Lysine requirement of growing male Pekin ducks.

    PubMed

    Bons, A; Timmler, R; Jeroch, H

    2002-12-01

    1. One growth experiment and one balance test were conducted to study the response to increasing levels of dietary lysine supplementation in male Pekin ducks with special reference to the growth periods from 1 to 3 weeks and 4 to 7 weeks of age. 2. Two different low-lysine diets were used as basal diets in both periods. The basal lysine levels were 7.6 g/kg (d 1 to 21) and 6.2 g/kg (d 22 to 49) and the ranges in lysine concentration were 7.6 to 12.6 g/kg (d 1 to 21) and 6.2 to 11.2 g/kg (d 22 to 49). 3. Growth performance, feed conversion efficiency and meat yield increased (P < 0.05) with increasing lysine concentration (requirement defined as 95% of the asymptote). 4. It is concluded that the dietary lysine concentration should be 0.93 g/MJ nitrogen corrected apparent metabolisable energy (AMEN) (11.7 g/kg) for the starter period (until d 21) and 0.75 g/MJ AMEN (10.0 g/kg) for the grower period (from d 22 onwards).

  14. Regulation of thrombosis and vascular function by protein methionine oxidation.

    PubMed

    Gu, Sean X; Stevens, Jeff W; Lentz, Steven R

    2015-06-18

    Redox biology is fundamental to both normal cellular homeostasis and pathological states associated with excessive oxidative stress. Reactive oxygen species function not only as signaling molecules but also as redox regulators of protein function. In the vascular system, redox reactions help regulate key physiologic responses such as cell adhesion, vasoconstriction, platelet aggregation, angiogenesis, inflammatory gene expression, and apoptosis. During pathologic states, altered redox balance can cause vascular cell dysfunction and affect the equilibrium between procoagulant and anticoagulant systems, contributing to thrombotic vascular disease. This review focuses on the emerging role of a specific reversible redox reaction, protein methionine oxidation, in vascular disease and thrombosis. A growing number of cardiovascular and hemostatic proteins are recognized to undergo reversible methionine oxidation, in which methionine residues are posttranslationally oxidized to methionine sulfoxide. Protein methionine oxidation can be reversed by the action of stereospecific enzymes known as methionine sulfoxide reductases. Calcium/calmodulin-dependent protein kinase II is a prototypical methionine redox sensor that responds to changes in the intracellular redox state via reversible oxidation of tandem methionine residues in its regulatory domain. Several other proteins with oxidation-sensitive methionine residues, including apolipoprotein A-I, thrombomodulin, and von Willebrand factor, may contribute to vascular disease and thrombosis.

  15. A 4-week toxicity study of methionine in male rats.

    PubMed

    Chin, Keigi; Toue, Sakino; Kawamata, Yasuko; Watanabe, Akiko; Miwa, Tadashi; Smriga, Miro; Sakai, Ryosei

    2015-01-01

    To examine 4-week toxicity of l-methionine (methionine), 5-week-old Fisher strain male rats were fed on diets containing 0, 0.1, 0.3, 0.9, 2.7 (w/w) of added methionine. Although no deaths were recorded, the highest dose of methionine (2.7% [w/w] of diet) reduced food intake and significantly suppressed growth rate. Growth suppression was characterized by an increase in hemolysis, splenic, and hepatic accumulation of hemosiderin, hemolytic anemia, and promotion of hematopoiesis. Other changes observed in the highest methionine intake group were a decrease in white blood cell count, thymus atrophy, and histological abnormalities in the adrenal gland and testis. Small, but significant, growth suppression, accompanied by some minor changes in plasma biochemical parameters, was also seen in rats fed on a test diet containing 0.9% (w/w) of additional methionine. Thus, no-observed-adverse-effect-level (NOAEL) and lowest-observed-adverse-effect level (LOAEL) of diet-added methionine were determined at 0.3% and 0.9% (w/w), corresponding to 236 and 705 mg/kg/d body weight, respectively. Since the basal diet contained protein-bound methionine at 0.5% (w/w), NOAEL and LOAEL of total dietary methionine were estimated at 0.8% and 1.4% (w/w) of diet.

  16. Mechanistic and Kinetic Study of Singlet O2 Oxidation of Methionine by On-Line Electrospray Ionization Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Liu, Fangwei; Lu, Wenchao; Yin, Xunlong; Liu, Jianbo

    2016-01-01

    We report a reaction apparatus developed to monitor singlet oxygen (1O2) reactions in solution using on-line ESI mass spectrometry and spectroscopy measurements. 1O2 was generated in the gas phase by the reaction of H2O2 with Cl2, detected by its emission at 1270 nm, and bubbled into aqueous solution continuously. 1O2 concentrations in solution were linearly related to the emission intensities of airborne 1O2, and their absolute scales were established based on a calibration using 9,10-anthracene dipropionate dianion as an 1O2 trapping agent. Products from 1O2 oxidation were monitored by UV-Vis absorption and positive/negative ESI mass spectra, and product structures were elucidated using collision-induced dissociation-tandem mass spectrometry. To suppress electrical discharge in negative ESI of aqueous solution, methanol was added to electrospray via in-spray solution mixing using theta-glass ESI emitters. Capitalizing on this apparatus, the reaction of 1O2 with methionine was investigated. We have identified methionine oxidation intermediates and products at different pH, and measured reaction rate constants. 1O2 oxidation of methionine is mediated by persulfoxide in both acidic and basic solutions. Persulfoxide continues to react with another methionine, yielding methionine sulfoxide as end-product albeit with a much lower reaction rate in basic solution. Density functional theory was used to explore reaction potential energy surfaces and establish kinetic models, with solvation effects simulated using the polarized continuum model. Combined with our previous study of gas-phase methionine ions with 1O2, evolution of methionine oxidation pathways at different ionization states and in different media is described.

  17. Mechanistic and Kinetic Study of Singlet O2 Oxidation of Methionine by On-Line Electrospray Ionization Mass Spectrometry.

    PubMed

    Liu, Fangwei; Lu, Wenchao; Yin, Xunlong; Liu, Jianbo

    2016-01-01

    We report a reaction apparatus developed to monitor singlet oxygen ((1)O2) reactions in solution using on-line ESI mass spectrometry and spectroscopy measurements. (1)O2 was generated in the gas phase by the reaction of H2O2 with Cl2, detected by its emission at 1270 nm, and bubbled into aqueous solution continuously. (1)O2 concentrations in solution were linearly related to the emission intensities of airborne (1)O2, and their absolute scales were established based on a calibration using 9,10-anthracene dipropionate dianion as an (1)O2 trapping agent. Products from (1)O2 oxidation were monitored by UV-Vis absorption and positive/negative ESI mass spectra, and product structures were elucidated using collision-induced dissociation-tandem mass spectrometry. To suppress electrical discharge in negative ESI of aqueous solution, methanol was added to electrospray via in-spray solution mixing using theta-glass ESI emitters. Capitalizing on this apparatus, the reaction of (1)O2 with methionine was investigated. We have identified methionine oxidation intermediates and products at different pH, and measured reaction rate constants. (1)O2 oxidation of methionine is mediated by persulfoxide in both acidic and basic solutions. Persulfoxide continues to react with another methionine, yielding methionine sulfoxide as end-product albeit with a much lower reaction rate in basic solution. Density functional theory was used to explore reaction potential energy surfaces and establish kinetic models, with solvation effects simulated using the polarized continuum model. Combined with our previous study of gas-phase methionine ions with (1)O2, evolution of methionine oxidation pathways at different ionization states and in different media is described.

  18. Nucleosome Binding Alters the Substrate Bonding Environment of Histone H3 Lysine 36 Methyltransferase NSD2.

    PubMed

    Poulin, Myles B; Schneck, Jessica L; Matico, Rosalie E; Hou, Wangfang; McDevitt, Patrick J; Holbert, Marc; Schramm, Vern L

    2016-06-01

    Nuclear receptor-binding SET domain protein 2 (NSD2) is a histone H3 lysine 36 (H3K36)-specific methyltransferase enzyme that is overexpressed in a number of cancers, including multiple myeloma. NSD2 binds to S-adenosyl-l-methionine (SAM) and nucleosome substrates to catalyze the transfer of a methyl group from SAM to the ε-amino group of histone H3K36. Equilibrium binding isotope effects and density functional theory calculations indicate that the SAM methyl group is sterically constrained in complex with NSD2, and that this steric constraint is released upon nucleosome binding. Together, these results show that nucleosome binding to NSD2 induces a significant change in the chemical environment of enzyme-bound SAM. PMID:27183271

  19. Using ‘biased-privileged’ scaffolds to identify lysine methyltransferase inhibitors

    PubMed Central

    Kashyap, Sudhir; Sandler, Joel; Martinez, Eduardo J.; Kapoor, Tarun M.

    2014-01-01

    Methylation of histones by lysine methyltransferases (KMTases) plays important roles in regulating chromatin function. It is also now clear that improper KMTases activity is linked to human diseases, such as cancer. We report an approach that employs drug-like ‘privileged’ scaffolds biased with motifs present in S-adenosyl methionine, the cofactor used by KMTases, to efficiently generate inhibitors for Set7, a biochemically well-characterized KMTase. Setin-1, the most potent inhibitor of Set7 we have developed also inhibits the KMTase G9a. Together these data suggest that these inhibitors should provide good starting points to generate useful probes for KMTase biology and guide the design of KMTase inhibitors with drug-like properties. PMID:24650704

  20. Free amino acid composition in hemolymph and muscle of the ghost crab, Ocypode platytarsis.

    PubMed

    Sankar, R Siva; Yogamoorthi, A

    2012-05-15

    Free amino acid plays an important role in physiological functions. The ghost crab, Ocypode platytarsis caught off the Puducherry sandy beaches, south east coast of India were investigated for the pattern of distribution of free amino acids. The hemolymph and muscle were sampled from male and female crabs and then subjected to free amino acid analysis quantitatively by HPLC. Fourteen free amino acids have been determined in hemolymph and muscle of both sexes in Ocypode platytarsis. The essential amino acids were leucine, lysine, methionine, phenylalanine, threonine, valine and histidine and non-essential amino acids were alanine, arginine, glutamic acid, glycine, serine and tyrosine. The total concentration of free amino acid found in the male and female hemolymph was lower than that in the muscle. Glutamic acid is of higher concentration in the hemolymph whereas histidine found was higher in the muscle of both sexes. The male crab contains a strikingly higher concentration of histidine of about eight times than that of female in muscles. The study revealed that distribution pattern of free amino acids differ in relation to hemolymph and muscle of male and female crabs.

  1. Gender differences in methionine accumulation and metabolism in freshly isolated mouse hepatocytes: Potential roles in toxicity

    SciTech Connect

    Dever, Joseph T.; Elfarra, Adnan A.

    2009-05-01

    L-Methionine (Met) is hepatotoxic at high concentrations. Because Met toxicity in freshly isolated mouse hepatocytes is gender-dependent, the goal of this study was to assess the roles of Met accumulation and metabolism in the increased sensitivity of male hepatocytes to Met toxicity compared with female hepatocytes. Male hepatocytes incubated with Met (30 mM) at 37 {sup o}C exhibited higher levels of intracellular Met at 0.5, 1.0, and 1.5 h, respectively, compared to female hepatocytes. Conversely, female hepatocytes had higher levels of S-adenosyl-L-methionine compared to male hepatocytes. Female hepatocytes also exhibited higher L-methionine-L-sulfoxide levels relative to control hepatocytes, whereas the increases in L-methionine-D-sulfoxide (Met-D-O) levels were similar in hepatocytes of both genders. Addition of aminooxyacetic acid (AOAA), an inhibitor of Met transamination, significantly increased Met levels at 1.5 h and increased Met-D-O levels at 1.0 and 1.5 h only in Met-exposed male hepatocytes. No gender differences in cytosolic Met transamination activity by glutamine transaminase K were detected. However, female mouse liver cytosol exhibited higher methionine-DL-sulfoxide (MetO) reductase activity than male mouse liver cytosol at low (0.25 and 0.5 mM) MetO concentrations. Collectively, these results suggest that increased cellular Met accumulation, decreased Met transmethylation, and increased Met and MetO transamination in male mouse hepatocytes may be contributing to the higher sensitivity of the male mouse hepatocytes to Met toxicity in comparison with female mouse hepatocytes.

  2. Substitution of lysine for arginine in the N-terminal 217th amino acid residue of the H gamma II of Staphylococcal gamma-hemolysin lowers the activity of the toxin.

    PubMed

    Sudo, K; Choorit, W; Asami, I; Kaneko, J; Muramoto, K; Kamio, Y

    1995-09-01

    The staphylococcal toxin gamma-hemolysin consists of two protein components, LukF and H gamma II. Staphylococcus aureus P83 was found to have five components, LukF, LukF-PV, LukM, LukS, and H gamma II for leukocidin or gamma-hemolysin. H gamma II of S. aureus P83 was demonstrated to be a naturally-occurring analogous molecule of H gamma II [H gamma II(P83)], in which the 217th arginine residue was replaced by lysine. The H gamma II(P83) showed about 50% of the hemolytic activity of normal H gamma II in the presence of LukF.

  3. Boron doped diamond and glassy carbon electrodes comparative study of the oxidation behaviour of cysteine and methionine.

    PubMed

    Enache, T A; Oliveira-Brett, A M

    2011-04-01

    The electrochemical oxidation behaviour at boron doped diamond and glassy carbon electrodes of the sulphur-containing amino acids cysteine and methionine, using cyclic and differential pulse voltammetry over a wide pH range, was compared. The oxidation reactions of these amino acids are irreversible, diffusion-controlled pH dependent processes, and occur in a complex cascade mechanism. The amino acid cysteine undergoes similar three consecutive oxidation reactions at both electrodes. The first step involves the oxidation of the sulfhydryl group with radical formation, that undergoes nucleophilic attack by water to give an intermediate species that is oxidized in the second step to cysteic acid. The oxidation of the sulfhydryl group leads to a disulfide bridge between two similar cysteine moieties forming cysteine. The subsequent oxidation of cystine occurs at a higher potential, due to the strong disulfide bridge covalent bond. The electro-oxidation of methionine at a glassy carbon electrode occurs in two steps, corresponding to the formation of sulfoxide and sulfone, involving the adsorption and protonation/deprotonation of the thiol group, followed by electrochemical oxidation. Methionine undergoes a one-step oxidation reaction at boron doped diamond electrodes due to the negligible adsorption, and the oxidation also leads to the formation of methionine sulfone. PMID:21377428

  4. The effect of replacement of methionine by homocystine on survival of malignant and normal adult mammalian cells in culture.

    PubMed

    Halpern, B C; Clark, B R; Hardy, D N; Halpern, R M; Smith, R A

    1974-04-01

    In tissue cultures of normal adult and malignant mammalian cells, homocystine has been substituted for methionine in a medium rich in folic acid and cyanocobalamin. Normal adult cells thrive. Three highly malignant cell types from three different species, including man, die.

  5. Identification of Methionine Sulfoxide Diastereomers in Immunoglobulin Gamma Antibodies Using Methionine Sulfoxide Reductase enzymes

    SciTech Connect

    Khor, Hui K.; Jacoby, Michael E.; Squier, Thomas C.; Chu, Grace C.; Chelius, Dirk

    2010-06-01

    During prolonged periods of storage methionines in antibodies and other proteins are known to become oxidized to form methionine sulfoxides and sulfones. While these post-translational modifications are commonly identified by peptide mapping, it is currently problematic to identify the relative abundances of the S- and R-diastereomers of methionine sulfoxide (Met(O)) due to their identical polarities and masses. Accordingly, we have developed a separation methodology for the rapid and quantitative determination of the relative abundances of Met(O) diastereomers. Identification of these diastereomers takes advantage of the complementary stereospecificities of methionine sulfoxide reductase (Msr) enzymes MsrA and MsrB, which respectively promote the selective reduction of S- and R-diastereomers of Met(O). In addition, an MsrBA fusion protein that contained both Msr enzyme activities permitted the quantitative reduction of all Met(O). Using these Msr enzymes in combination with peptide mapping we are able to detect and differentiate Met-diastereomers in a monoclonal IgG2 and IgG1 antibody. We also monitored the formation of sulfones and studied the rate of oxidation in the different Met residues in our IgG2 antibody. The reported ability to separate and identify diastereomers of Met(O) permits a more complete characterization of Met oxidation products. All the affected Met residues (M251, M427, M396) in this study are conserved in human IgG sequences and therefore offer predictive potential in characterizing oxidative modification.

  6. Pyridinylpyrimidines selectively inhibit human methionine aminopeptidase-1.

    PubMed

    Zhang, Pengtao; Yang, Xinye; Zhang, Feiran; Gabelli, Sandra B; Wang, Renxiao; Zhang, Yihua; Bhat, Shridhar; Chen, Xiaochun; Furlani, Manuel; Amzel, L Mario; Liu, Jun O; Ma, Dawei

    2013-05-01

    Cellular protein synthesis is initiated with methionine in eukaryotes with few exceptions. Methionine aminopeptidases (MetAPs) which catalyze the process of N-terminal methionine excision are essential for all organisms. In mammals, type 2 MetAP (MetAP2) is known to be important for angiogenesis, while type 1 MetAP (MetAP1) has been shown to play a pivotal role in cell proliferation. Our previous high-throughput screening of a commercial compound library uncovered a novel class of inhibitors for both human MetAP1 (HsMetAP1) and human MetAP2 (HsMetAP2). This class of inhibitors contains a pyridinylpyrimidine core. To understand the structure-activity relationship (SAR) and to search for analogues of 2 with greater potency and higher HsMetAP1-selectivity, a total of 58 analogues were acquired through either commercial source or by in-house synthesis and their inhibitory activities against HsMetAP1 and HsMetAP2 were determined. Through this systematic medicinal chemistry analysis, we have identified (1) 5-chloro-6-methyl-2-pyridin-2-ylpyrimidine as the minimum element for the inhibition of HsMetAP1; (2) 5'-chloro as the favored substituent on the pyridine ring for the enhanced potency against HsMetAP1; and (3) long C4 side chains as the essentials for higher HsMetAP1-selectivity. At the end of our SAR campaign, 25b, 25c, 26d and 30a-30c are among the most selective and potent inhibitors of purified HsMetAP1 reported to date. In addition, we also performed crystallographic analysis of one representative inhibitor (26d) in complex with N-terminally truncated HsMetAP1.

  7. Nutritional and medicinal aspects of D-amino acids.

    PubMed

    Friedman, Mendel; Levin, Carol E

    2012-05-01

    This paper reviews and interprets a method for determining the nutritional value of D-amino acids, D-peptides, and amino acid derivatives using a growth assay in mice fed a synthetic all-amino acid diet. A large number of experiments were carried out in which a molar equivalent of the test compound replaced a nutritionally essential amino acid such as L-lysine (L-Lys), L-methionine (L-Met), L-phenylalanine (L-Phe), and L-tryptophan (L-Trp) as well as the semi-essential amino acids L-cysteine (L-Cys) and L-tyrosine (L-Tyr). The results show wide-ranging variations in the biological utilization of test substances. The method is generally applicable to the determination of the biological utilization and safety of any amino acid derivative as a potential nutritional source of the corresponding L-amino acid. Because the organism is forced to use the D-amino acid or amino acid derivative as the sole source of the essential or semi-essential amino acid being replaced, and because a free amino acid diet allows better control of composition, the use of all-amino-acid diets for such determinations may be preferable to protein-based diets. Also covered are brief summaries of the widely scattered literature on dietary and pharmacological aspects of 27 individual D-amino acids, D-peptides, and isomeric amino acid derivatives and suggested research needs in each of these areas. The described results provide a valuable record and resource for further progress on the multifaceted aspects of D-amino acids in food and biological samples.

  8. L-methionine degradation potentialities of cheese-ripening microorganisms.

    PubMed

    Bonnarme, P; Lapadatescu, C; Yvon, M; Spinnler, H E

    2001-11-01

    Volatile sulphur compounds are major flavouring compounds in many traditional fermented foods including cheeses. These compounds are products of the catabolism of L-methionine by cheese-ripening microorganisms. The diversity of L-methionine degradation by such microorganisms, however, remains to be characterized. The objective of this work was to compare the capacities to produce volatile sulphur compounds by five yeasts, Geotrichum candidum, Yarrowia lipolytica, Kluyveromyces lactis, Debaryomyces hansenii, Saccharomyces cerevisiae and five bacteria, Brevibacterium linens, Corynebacterium glutamicum, Arthrobacter sp., Micrococcus lutens and Staphylococcus equorum of technological interest for cheese-ripening. The ability of whole cells of these microorganisms to generate volatile sulphur compounds from L-methionine was compared. The microorganisms produced a wide spectrum of sulphur compounds including methanethiol, dimethylsulfide, dimethyldisulfide, dimethyltrisulfide and also S-methylthioesters, which varied in amount and type according to strain. Most of the yeasts produced methanethiol, dimethylsulfide, dimethyldisulfide and dimethyltrisulfide but did not produce S-methylthioesters, apart from G. candidum that produced S-methyl thioacetate. Bacteria, especially Arth. sp. and Brevi. linens, produced the highest amounts and the greatest variety of volatile sulphur compounds includling methanethiol, sulfides and S-methylthioesters, e.g. S-methyl thioacetate, S-methyl thiobutyrate, S-methyl thiopropionate and S-methyl thioisovalerate. Cell-free extracts of all the yeasts and bacteria were examined for the activity of enzymes possibly involved in L-methionine catabolism, i.e. L-methionine demethiolase, L-methionine aminotransferase and L-methionine deaminase. They all possessed L-methionine demethiolase activity, while some (K. lactis, Deb. hansenii, Arth. sp., Staph. equorum) were deficient in L-methionine aminotransferase, and none produced L-methionine deaminase

  9. Simultaneous analysis of Nε-(carboxymethyl)lysine, reducing sugars, and lysine during the dairy thermal process.

    PubMed

    Xu, Xian-Bing; Ma, Fei; Yu, Shu-Juan; Guan, Yong-Guang

    2013-09-01

    A new analytical method allowing the simultaneous quantification of Nε-(carboxymethyl)lysine (CML), lysine, and reducing sugars (glucose, lactose, and galactose) is described. It is based on high performance anion-exchange chromatography with pulsed amperometric electrochemical detection. This method demonstrated a low limit of quantification (0.385 to 0.866 mg/L), excellent linear correlation (R(2)>0.997), and desired calibration range (3.125 to 25 mg/L). In addition, lactose-lysine solutions containing sulfite (4 to 400 mmol/L) were heated at 110°C for 2h. The results showed that sulfite inhibited the formation of CML and promoted the consumption of reducing sugars and lysine in the Maillard reaction model. The method proved to be useful for simultaneous analysis of CML, lysine, and reducing sugars (glucose, galactose, and lactose) in the Maillard reaction system. Moreover, sulfite was an effective inhibitor of CML formation.

  10. Chromatographic behavior of peptides containing oxidized methionine residues in proteomic LC-MS experiments: Complex tale of a simple modification.

    PubMed

    Lao, Ying W; Gungormusler-Yilmaz, Mine; Shuvo, Sabbir; Verbeke, Tobin; Spicer, Vic; Krokhin, Oleg V

    2015-07-01

    On average, the oxidation of a single Met residue to Mso (methionine S-oxide, methionine sulfoxide) and Msn (methionine S,S-dioxide, methionine sulfone) decreases peptide retention in RP HPLC by 2.37 and 1.95 Hydrophobicity Index units (% acetonitrile), respectively. At the same time, the magnitude of the retention shift varies greatly (-9.1 to +0.4% acetonitrile for Mso) depending on peptide sequence. The latter effects are mostly associated with the stabilization of secondary structures upon peptide interaction with the hydrophobic stationary phase: when an oxidized residue is located in the hydrophobic face of an amphipathic helix, the decrease in retention is profound. The same amino acid positioning leads to complete or partial resolution of pairs of peptides containing diastereomeric Mso residues. Contrary to all previously reported observations, and the nature of this modification, we also demonstrate for the first time that methionine oxidation may increase peptide hydrophobicity. This behavior is characteristic for Met residues in the N3 position of an N-capping box stabilization motif prior to the amphipathic helix. All these findings indicate that the prediction of peptide secondary structures upon interaction with hydrophobic surfaces must become an integral part of peptide retention modeling in proteomic applications going forward.

  11. Effects of diet supplementation with white tea and methionine on lipid metabolism of gilthead sea bream juveniles (Sparus aurata).

    PubMed

    Pérez-Jiménez, Amalia; Peres, Helena; Rubio, Vera Cruz; Oliva-Teles, Aires

    2013-06-01

    A growth trial was performed with gilthead sea bream juveniles (Sparus aurata) to evaluate the effect of diet supplementation with white tea and methionine on fish performance and lipid metabolism. For that purpose, four diets were formulated: a fish meal-based diet (Control) and diets identical to the control diet but supplemented with 2.9 % white tea (Tea), 0.3 % methionine (Met) or 2.9 % white tea plus 0.3 % methionine (Tea + Met). Growth performance and feed efficiency parameters, whole-body and liver composition, plasma metabolites concentration and liver glucose 6-phosphate dehydrogenase (G6PDH), malic enzyme (ME) and fatty acid synthetase (FAS) activities were determined. Feed intake was higher in fish fed methionine-supplemented diets, whereas this parameter and growth was decreased in fish fed white tea supplementation. Feed efficiency and protein efficiency ratio were not affected by diet composition. Plasma HDL cholesterol and total lipids concentration were higher in fish fed white tea-supplemented diets. Whole-body lipid, plasma glucose, liver glycogen concentration and liver G6PDH, ME and FAS activities were lower in fish fed white tea-supplemented diets. Results of the present study indicate that methionine seems to act as a feed attractant in diets for sea bream juveniles. Additionally, white tea is an important modulator of lipid metabolism in sea bream juveniles.

  12. Dependence on exogenous methionine of rat sarcoma and murine leukemia cells in culture.

    PubMed

    Koziorowska, J; Pieńkowska, K; Tautt, J

    1980-01-01

    A comparative study was performed on methionine auxotrophy of rat sarcoma and murine leukemia cells taken directly from the organism and grown in culture in media lacking methionine or in which methionine was substituted by homocysteine. Methionine auxotrophy was observed in both kinds of cells. At low levels of methionine in the media containing homocysteine rat sarcoma cells showed an increase in growth. Addition of homocysteine to the media with low levels of methionine did not influence the survival of murine leukemia cells.

  13. Child Stunting is Associated with Low Circulating Essential Amino Acids

    PubMed Central

    Semba, Richard D.; Shardell, Michelle; Sakr Ashour, Fayrouz A.; Moaddel, Ruin; Trehan, Indi; Maleta, Kenneth M.; Ordiz, M. Isabel; Kraemer, Klaus; Khadeer, Mohammed A.; Ferrucci, Luigi; Manary, Mark J.

    2016-01-01

    Background Stunting affects about one-quarter of children under five worldwide. The pathogenesis of stunting is poorly understood. Nutritional interventions have had only modest effects in reducing stunting. We hypothesized that insufficiency in essential amino acids may be limiting the linear growth of children. Methods We used a targeted metabolomics approach to measure serum amino acids, glycerophospholipids, sphingolipids, and other metabolites using liquid chromatography-tandem mass spectrometry in 313 children, aged 12–59 months, from rural Malawi. Children underwent anthropometry. Findings Sixty-two percent of the children were stunted. Children with stunting had lower serum concentrations of all nine essential amino acids (tryptophan, isoleucine, leucine, valine, methionine, threonine, histidine, phenylalanine, lysine) compared with nonstunted children (p < 0.01). In addition, stunted children had significantly lower serum concentrations of conditionally essential amino acids (arginine, glycine, glutamine), non-essential amino acids (asparagine, glutamate, serine), and six different sphingolipids compared with nonstunted children. Stunting was also associated with alterations in serum glycerophospholipid concentrations. Interpretation Our findings support the idea that children with a high risk of stunting may not be receiving an adequate dietary intake of essential amino acids and choline, an essential nutrient for the synthesis of sphingolipids and glycerophospholipids. PMID:27211567

  14. Plasma concentrations and intakes of amino acids in male meat-eaters, fish-eaters, vegetarians and vegans: a cross-sectional analysis in the EPIC-Oxford cohort

    PubMed Central

    Schmidt, J A; Rinaldi, S; Scalbert, A; Ferrari, P; Achaintre, D; Gunter, M J; Appleby, P N; Key, T J; Travis, R C

    2016-01-01

    Background/Objectives: We aimed to investigate the differences in plasma concentrations and in intakes of amino acids between male meat-eaters, fish-eaters, vegetarians and vegans in the Oxford arm of the European Prospective Investigation into Cancer and Nutrition. Subjects/Methods: This cross-sectional analysis included 392 men, aged 30–49 years. Plasma amino acid concentrations were measured with a targeted metabolomic approach using mass spectrometry, and dietary intake was assessed using a food frequency questionnaire. Differences between diet groups in mean plasma concentrations and intakes of amino acids were examined using analysis of variance, controlling for potential confounding factors and multiple testing. Results: In plasma, concentrations of 6 out of 21 amino acids varied significantly by diet group, with differences of −13% to +16% between meat-eaters and vegans. Concentrations of methionine, tryptophan and tyrosine were highest in fish-eaters and vegetarians, followed by meat-eaters, and lowest in vegans. A broadly similar pattern was seen for lysine, whereas alanine concentration was highest in fish-eaters and lowest in meat-eaters. For glycine, vegans had the highest concentration and meat-eaters the lowest. Intakes of all 18 dietary amino acids differed by diet group; for the majority of these, intake was highest in meat-eaters followed by fish-eaters, then vegetarians and lowest in vegans (up to 47% lower than in meat-eaters). Conclusions: Men belonging to different habitual diet groups have significantly different plasma concentrations of lysine, methionine, tryptophan, alanine, glycine and tyrosine. However, the differences in plasma concentrations were less marked than and did not necessarily mirror those seen for amino acid intakes. PMID:26395436

  15. Complete assignment of the methionyl carbonyl carbon resonance in switch variant anti-dansyl antibodies labeled with (1- sup 13 C)methionine

    SciTech Connect

    Kato, Koichi; Matsunaga, C.; Igarashi, Takako; Kim, Hahyung; Odaka, Asano; Shimada, Ichio; Arata, Yoji )

    1991-01-01

    A {sup 13}C NMR study is reported of switch variant anti-dansyl antibodies developed by Dangl et al. who had used the fluorescence-activated cell sorter to select and clone these variants. These switch variant antibodies possess the identical V{sub H}, V{sub L}, and C{sub L} domains in conjunction with different heavy chain constant regions. In the present study, switch variant antibodies of IgG1, IgG2a, and IgG2b subclasses were used along with a short-chain IgG2a antibody, in which the entire C{sub H}1 domain is deleted. The switch variant antibodies were specifically labeled with (1-{sup 13}C)methionine by growing hybridoma cells in serum-free medium. Assignments of all the methionyl carbonyl carbon resonances have been completed by using the intact antibodies along with their fragments and recombined proteins in which either heavy or light chain is labeled. A double labeling method has played a crucial role in the process of the spectral assignments. The strategy used for the assignments has been described in detail. In incorporating {sup 15}N-labeled amino acids into the antibodies for the double labeling, isotope dilution caused a serious problem except in the cases of ({alpha}-{sup 15}N)lysine and ({sup 15}N)threonine, both of which cannot become the substrate of transaminases. It was found that {beta}-chloro-L-alanine is most effective in suppressing the isotope scrambling. So far, spectral assignments by the double labeling method have been possible with {sup 15}N-labeled Ala, His, Ile, Lys, Met, Ser, Thr, Tyr, and Val. On the basis of the results of the present {sup 13}C study, possible use of the assigned carbonyl carbon resonances for the elucidation of the structure-function relationship in the antibody system has been briefly discussed.

  16. A Method to Determine Lysine Acetylation Stoichiometries

    DOE PAGES

    Nakayasu, Ernesto S.; Wu, Si; Sydor, Michael A.; Shukla, Anil K.; Weitz, Karl K.; Moore, Ronald J.; Hixson, Kim K.; Kim, Jong-Seo; Petyuk, Vladislav A.; Monroe, Matthew E.; et al

    2014-01-01

    Lysine acetylation is a common protein posttranslational modification that regulates a variety of biological processes. A major bottleneck to fully understanding the functional aspects of lysine acetylation is the difficulty in measuring the proportion of lysine residues that are acetylated. Here we describe a mass spectrometry method using a combination of isotope labeling and detection of a diagnostic fragment ion to determine the stoichiometry of protein lysine acetylation. Using this technique, we determined the modification occupancy for ~750 acetylated peptides from mammalian cell lysates. Furthermore, the acetylation on N-terminal tail of histone H4 was cross-validated by treating cells with sodiummore » butyrate, a potent deacetylase inhibitor, and comparing changes in stoichiometry levels measured by our method with immunoblotting measurements. Of note we observe that acetylation stoichiometry is high in nuclear proteins, but very low in mitochondrial and cytosolic proteins. In summary, our method opens new opportunities to study in detail the relationship of lysine acetylation levels of proteins with their biological functions.« less

  17. Crystal Structure of Ll-Diaminopimelate Aminotransferase From 'Arabidopsis Thaliana': a Recently-Discovered Enzyme in the Biosynthesis of L-Lysine By Plants And 'Chlamydia'

    SciTech Connect

    Watanabe, N.; Cherney, M.M.; van Belkum, M.J.; Marcus, S.L.; Flegel, M.D.; Clay, M.D.; Deyholos, M.K.; Vederas, J.C.; James, M.N.G.

    2007-07-13

    The essential biosynthetic pathway to l-Lysine in bacteria and plants is an attractive target for the development of new antibiotics or herbicides because it is absent in humans, who must acquire this amino acid in their diet. Plants use a shortcut of a bacterial pathway to l-Lysine in which the pyridoxal-5-phosphate (PLP)-dependent enzyme ll-diaminopimelate aminotransferase (LL-DAP-AT) transforms l-tetrahydrodipicolinic acid (L-THDP) directly to LL-DAP. In addition, LL-DAP-AT was recently found in Chlamydia sp., suggesting that inhibitors of this enzyme may also be effective against such organisms. In order to understand the mechanism of this enzyme and to assist in the design of inhibitors, the three-dimensional crystal structure of LL-DAP-AT was determined at 1.95 Angstroms resolution. The cDNA sequence of LL-DAP-AT from Arabidopsis thaliana (AtDAP-AT) was optimized for expression in bacteria and cloned in Escherichia coli without its leader sequence but with a C-terminal hexahistidine affinity tag to aid protein purification. The structure of AtDAP-AT was determined using the multiple-wavelength anomalous dispersion (MAD) method with a seleno-methionine derivative. AtDAP-AT is active as a homodimer with each subunit having PLP in the active site. It belongs to the family of type I fold PLP-dependent enzymes. Comparison of the active site residues of AtDAP-AT and aspartate aminotransferases revealed that the PLP binding residues in AtDAP-AT are well conserved in both enzymes. However, Glu97* and Asn309* in the active site of AtDAP-AT are not found at similar positions in aspartate aminotransferases, suggesting that specific substrate recognition may require these residues from the other monomer. A malate-bound structure of AtDAP-AT allowed LL-DAP and L-glutamate to be modeled into the active site. These initial three-dimensional structures of LL-DAP-AT provide insight into its substrate specificity and catalytic mechanism.

  18. Methionine as a Precursor of Ethylene—Commentary

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lieberman et al. showed in a 1966 publication of Plant Physiology that methionine is a precursor of ethylene. It was the first paper that showed ethylene carbons are derived from carbons 3 and 4 of methionine. This paper catalyzed remarkable interest among plant biologists to elucidate the biosynth...

  19. High Methionine Diet Poses Cardiac Threat: A Molecular Insight.

    PubMed

    Chaturvedi, Pankaj; Kamat, Pradip K; Kalani, Anuradha; Familtseva, Anastasia; Tyagi, Suresh C

    2016-07-01

    High methionine diet (HMD) for example red meat which includes lamb, beef, pork can pose cardiac threat and vascular dysfunction but the mechanisms are unclear. We hypothesize that a diet rich in methionine can malfunction the cardiovascular system in three ways: (1) by augmenting oxidative stress; (2) by inflammatory manifestations; and (3) by matrix/vascular remodeling. To test this hypothesis we used four groups of mice: (1) WT; (2) WT + methionine; (3) CBS(+/-) ; (4) CBS(+/-) +methionine. We observed high oxidative stress in mice fed with methionine which was even higher in CBS(+/-) and CBS(+/-) +methionine. Higher oxidative stress was indicated by high levels of SOD-1 in methionine fed mouse hearts whereas IL-1β, IL-6, TNFα, and TLR4 showed high inflammatory manifestations. The upregulated levels of eNOS/iNOS and upregulated levels of MMP2/MMP9 along with high collagen deposition indicated vascular and matrix remodeling in methionine fed mouse. We evaluated the cardiac function which was dysregulated in the mice fed with HMD. These mice had decreased ejection fraction and left ventricular dysfunction which subsequently leads to adverse cardiac remodeling. In conclusion, our study clearly shows that HMD poses a cardiac threat by increasing oxidative stress, inflammatory manifestations, matrix/vascular remodeling, and decreased cardiac function.

  20. Effect of dietary lysine on growth, intestinal enzymes activities and antioxidant status of sub-adult grass carp (Ctenopharyngodon idella).

    PubMed

    Li, Xue-Yin; Tang, Ling; Hu, Kai; Liu, Yang; Jiang, Wei-Dan; Jiang, Jun; Wu, Pei; Chen, Gang-Fu; Li, Shu-Hong; Kuang, Sheng-Yao; Feng, Lin; Zhou, Xiao-Qiu

    2014-06-01

    The dietary lysine requirement of sub-adult grass carp (460 ± 1.5 g) was assessed by feeding diets supplemented with grade levels of lysine (6.6, 8.5, 10.8, 12.9, 15.0 and 16.7 g kg(-1) diet) for 56 days. The test diets (28% CP) contained fish meal, casein and gelatin as sources of intact protein, supplemented with crystalline amino acids. Weight gain (WG), feed intake and feed efficiency were significantly improved with increasing levels of lysine up to 12.9 g kg(-1) diet and thereafter declined (P < 0.05). Quadratic regression analysis of WG at 95% maximum response indicated lysine requirement was 10.9 g kg(-1) diet. Activities of trypsin, chymotrypsin, lipase, Na(+), K(+)-ATPase and alkaline phosphatase in intestine, creatine kinase activity in proximal and mid-intestine responded similar to WG (P < 0.05). In addition, lipid and protein oxidation decreased with increasing levels of lysine up to certain values and increased thereafter (P < 0.05); the anti-hydroxyl radical capacity, dismutase, catalase, glutathione peroxidase, glutathione reductase, glutathione-S-transferase (GST) activities and glutathione content were increased with increasing dietary lysine levels up to certain values in the detected tissues, except for hepatopancreatic GST. Requirement estimated on the basis of malondialdehyde content in intestine and hepatopancreas was 10.6 and 9.53 g lysine kg(-1) diet, respectively. PMID:24174167

  1. Enhanced L-lysine production from pretreated beet molasses by engineered Escherichia coli in fed-batch fermentation.

    PubMed

    He, Xun; Chen, Kequan; Li, Yan; Wang, Zhen; Zhang, Hong; Qian, Juan; Ouyang, Pingkai

    2015-08-01

    Faster sugar consumption rate and low-cost nitrogen source are required for the chemical biosynthesis using molasses. Five pretreatment methods were applied to beet molasses prior to fermentation through engineered Escherichia coli, respectively, and corn steep liquid was used as an organic nitrogen source to replace expensive yeast extract. Furthermore, the effects of different feeding strategy in fed-batch fermentation on L-lysine production were investigated. The experimental results showed that combined tricalcium phosphate, sulfuric acid, and activated carbon pretreatment method (TPSA) pretreatment could improve the sugar consumption rate most greatly, and the initial total sugar concentration of 35 g/L from TPSA-pretreated beet molasses gave the best results with respect to L-lysine production, dry cell weight concentration, and L-lysine yield in batch fermentation. Moreover, a mixture of low-cost corn steep liquid and yeast extract containing equal amount of nitrogen could be used as the organic nitrogen source for effective L-lysine fermentation, and constant speed feeding strategy of TPSA-pretreated beet molasses promoted L-lysine production by engineered E. coli. The TPSA-pretreated beet molasses had a sugar consumption rate of 1.75 g/(L h), and a L-lysine yield of 27.81% was achieved, compared with the theoretical yield of 62% by glucose. It was clarified that the pretreatment significantly enhanced the conversion of sugars in beet molasses to L-lysine.

  2. Two protein lysine methyltransferases methylate outer membrane protein B from Rickettsia.

    PubMed

    Abeykoon, Amila H; Chao, Chien-Chung; Wang, Guanghui; Gucek, Marjan; Yang, David C H; Ching, Wei-Mei

    2012-12-01

    Rickettsia prowazekii, the etiologic agent of epidemic typhus, is a potential biological threat agent. Its outer membrane protein B (OmpB) is an immunodominant antigen and plays roles as protective envelope and as adhesins. The observation of the correlation between methylation of lysine residues in rickettsial OmpB and bacterial virulence has suggested the importance of an enzymatic system for the methylation of OmpB. However, no rickettsial lysine methyltransferase has been characterized. Bioinformatic analysis of genomic DNA sequences of Rickettsia identified putative lysine methyltransferases. The genes of the potential methyltransferases were synthesized, cloned, and expressed in Escherichia coli, and expressed proteins were purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. The methyltransferase activities of the purified proteins were analyzed by methyl incorporation of radioactively labeled S-adenosylmethionine into recombinant fragments of OmpB. Two putative recombinant methyltransferases (rRP789 and rRP027-028) methylated recombinant OmpB fragments. The specific activity of rRP789 is 10- to 30-fold higher than that of rRP027-028. Western blot analysis using specific antibodies against trimethyl lysine showed that both rRP789 and rRP027-028 catalyzed trimethylation of recombinant OmpB fragments. Liquid chromatography-tandem mass spectrometry (LC/MS-MS) analysis showed that rRP789 catalyzed mono-, di-, and trimethylation of lysine, while rRP027-028 catalyzed exclusively trimethylation. To our knowledge, rRP789 and rRP027-028 are the first biochemically characterized lysine methyltransferases of outer membrane proteins from Gram-negative bacteria. The production and characterization of rickettsial lysine methyltransferases provide new tools to investigate the mechanism of methylation of OmpB, effects of methylation on the structure and function of OmpB, and development of methylated OmpB-based diagnostic assays and vaccine candidates.

  3. Alimentary proteins, amino acids and cholesterolemia.

    PubMed

    Blachier, François; Lancha, Antonio H; Boutry, Claire; Tomé, Daniel

    2010-01-01

    Numerous data from both epidemiological and experimental origins indicate that some alimentary proteins and amino acids in supplements can modify the blood LDL cholesterol, HDL cholesterol and total cholesterol. After an initial approval of the health claim for soy protein consumption for the prevention of coronary heart disease, more recently it has been concluded from an overall analysis of literature that isolated soy protein with isoflavones only slightly decrease LDL and total cholesterol. Other plant extracts and also some proteins from animal origin have been reported to exert a lowering effect on blood cholesterol when compared with a reference protein (often casein). The underlying mechanisms are still little understood. Individual amino acids and mixture of amino acids have also been tested (mostly in animal studies) for their effects on cholesterol parameters and on cholesterol metabolism. Methionine, lysine, cystine, leucine, aspartate and glutamate have been tested individually and in combination in different models of either normo or hypercholesterolemic animals and found to be able to modify blood cholesterol and/or LDL cholesterol and/or HDL cholesterol. It is however not known if these results are relevant to human nutrition.

  4. Advances in Bacterial Methionine Aminopeptidase Inhibition.

    PubMed

    Helgren, Travis R; Wangtrakuldee, Phumvadee; Staker, Bart L; Hagen, Timothy J

    2016-01-01

    Methionine aminopeptidases (MetAPs) are metalloenzymes that cleave the N-terminal methionine from newly synthesized peptides and proteins. These MetAP enzymes are present in bacteria, and knockout experiments have shown that MetAP activity is essential for cell life, suggesting that MetAPs are good antibacterial drug targets. MetAP enzymes are also present in the human host and selectivity is essential. There have been significant structural biology efforts and over 65 protein crystal structures of bacterial MetAPs are deposited into the PDB. This review highlights the available crystallographic data for bacterial MetAPs. Structural comparison of bacterial MetAPs with human MetAPs highlights differences that can lead to selectivity. In addition, this review includes the chemical diversity of molecules that bind and inhibit the bacterial MetAP enzymes. Analysis of the structural biology and chemical space of known bacterial MetAP inhibitors leads to a greater understanding of this antibacterial target and the likely development of potential antibacterial agents.

  5. Structure and function of the methionine aminopeptidases.

    PubMed

    Lowther, W T; Matthews, B W

    2000-03-01

    The removal of the N-terminal methionine from proteins and peptides is dependent upon a novel class of proteases typified by the dinuclear metalloenzyme methionine aminopeptidase from Escherichia coli (eMetAP). Substantial progress has recently been made in determining the structures of several members of this family. The identification of human MetAP as the target of putative anti-cancer drugs reiterates the importance of this family of enzymes. Determination of the modes of binding to E. coli MetAP of a substrate-like bestatin-based inhibitor, as well as phosphorus-containing transition-state analogs and reaction products has led to a rationalization of the substrate specificity and suggested the presumed catalytic mechanism. The conservation of key active site residues and ligand interactions between the MetAPs and other enzyme of the same fold suggest that avoidance of cross-reactivity may be an important consideration in the design of inhibitors directed toward a single member of the family. PMID:10708856

  6. Structure and Mechanisms of Lysine Methylation Recognition by the Chromodomain in Gene Transcription†

    PubMed Central

    Yap, Kyoko L.; Zhou, Ming-Ming

    2011-01-01

    Histone methylation recognition is accomplished by a number of evolutionarily conserved protein domains, including those belonging to the methylated lysine-binding Royal family of structural folds. One well-known member of the Royal family, the chromodomain, is found in the HP1/Chromobox and CHD subfamilies of proteins, in addition to a small number of other proteins that are involved in chromatin remodeling and gene transcriptional silencing. Here we discuss the structure and function of the chromodomain within these proteins as histone methylated lysine binders, and how the functions of these chromodomains can be modulated by additional post-translational modifications or binding to nucleic acids. PMID:21288002

  7. Kinetics of microbial methionine metabolism in continuous cultures administered different methionine sources.

    PubMed

    Firkins, J L; Fowler, C M; Devillard, E; Bequette, B J

    2015-02-01

    The Met precursor 2-hydroxy-4-(methylthio) butanoic acid (HMB) is expected to be more extensively degraded in the rumen than its isopropyl ester (HMBi). A control and 3 isomolar treatments-0.097% dl-methionine, 0.11% HMBi (HMBi), and 0.055% HMBi plus 0.048% Met (Met + HMBi)-were dosed every 8h simultaneously with 3-times-daily feeding into continuous cultures. Starting on d 9, for 6 consecutive doses, both [1-(13)C]-l-Met and [methyl-(2)H3]-l-Met replaced part of the unlabeled dl-Met, [(13)C5]-dl-HMBi replaced a portion of the unlabeled dl-HMBi, and [1-(13)C]-l-Met plus [(13)C5]-dl-HMBi replaced a portion of the respective unlabeled doses for the Met + HMBi treatment. After the sixth dose (d 11), unlabeled Met or HMBi provided 100% of the doses to follow elimination kinetics of the labels in HMBi, free Met, and bacterial Met compartments. The free [1-(13)C]-l-Met recycled more and was recovered in bacterial Met to a lesser extent than was the free [methyl-(2)H3]-l-Met recycling and that was recovered in bacterial Met. Increasing HMBi inclusion (0, 50, and 100% substitution of the exogenously dosed Met on a molar equivalent basis) tended to increase HMBi escape from 54.7 to 71.3% for the 50 and 100% HMBi treatments, respectively. Despite HMBi substituting for and decreasing the dosage of Met, increasing HMBi increased accumulation of free Met in fermenter fluid. The HMBi (after de-esterification of the isopropyl group) presumably produces Met through the intermediate α-ketomethylthyiobutyrate with an aminotransferase that also has high affinity for branched-chain AA. We provide evidence that the HMBi-derived Met is likely released from bacterial cells and accumulates rather than being degraded, potentially as a result of lagging d-stereoisomer metabolism. More research is needed to evaluate racemization and metabolism of stereoisomers of HMBi, Met, and other AA in ruminal microbes.

  8. Brain uptake of ketoprofen-lysine prodrug in rats.

    PubMed

    Gynther, Mikko; Jalkanen, Aaro; Lehtonen, Marko; Forsberg, Markus; Laine, Krista; Ropponen, Jarmo; Leppänen, Jukka; Knuuti, Johanna; Rautio, Jarkko

    2010-10-31

    The blood-brain barrier (BBB) controls the entry of xenobiotics into the brain. Often the development of central nervous system drugs needs to be terminated because of their poor brain uptake. We describe a way to achieve large neutral amino acid transporter (LAT1)-mediated drug transport into the rat brain. We conjugated ketoprofen to an amino acid l-lysine so that the prodrug could access LAT1. The LAT1-mediated brain uptake of the prodrug was demonstrated with in situ rat brain perfusion technique. The ability of the prodrug to deliver ketoprofen into the site of action, the brain intracellular fluid, was determined combining in vivo and in vitro experiments. A rapid brain uptake from blood and cell uptake was seen both in in situ and in vivo experiments. Therefore, our results show that a prodrug approach can achieve uptake of drugs via LAT1 into the brain intracellular fluid. The distribution of the prodrug in the brain parenchyma and the site of parent drug release in the brain were shown with in vivo and in vitro studies. In addition, our results show that although lysine or ketoprofen are not LAT1-substrates themselves, by combining these molecules, the formed prodrug has affinity for LAT1. PMID:20727958

  9. Mycobacteriophage Lysin B is a novel mycolylarabinogalactan esterase

    SciTech Connect

    Payne, K.; Sun, Q.; Sacchettini, J.; Hatfull, G.F.

    2010-08-27

    Mycobacteriophages encounter a unique problem among phages of Gram-positive bacteria, in that lysis must not only degrade the peptidoglycan layer but also circumvent a mycolic acid-rich outer membrane covalently attached to the arabinogalactan-peptidoglycan complex. Mycobacteriophages accomplish this by producing two lysis enzymes, Lysin A (LysA) that hydrolyses peptidoglycan, and Lysin B (LysB), a novel mycolylarabinogalactan esterase, that cleaves the mycolylarabinogalactan bond to release free mycolic acids. The D29 LysB structure shows an {alpha}/{beta} hydrolase organization with a catalytic triad common to cutinases, but which contains an additional four-helix domain implicated in the binding of lipid substrates. Whereas LysA is essential for mycobacterial lysis, a Giles {Delta}lysB mutant mycobacteriophage is viable, but defective in the normal timing, progression and completion of host cell lysis. We propose that LysB facilitates lysis by compromising the integrity of the mycobacterial outer membrane linkage to the arabinogalactan-peptidoglycan layer.

  10. Elongation factor 1 alpha concentration is highly correlated with the lysine content of maize endosperm.

    PubMed Central

    Habben, J E; Moro, G L; Hunter, B G; Hamaker, B R; Larkins, B A

    1995-01-01

    Lysine is the most limiting essential amino acid in cereals, and for many years plant breeders have attempted to increase its concentration to improve the nutritional quality of these grains. The opaque2 mutation in maize doubles the lysine content in the endosperm, but the mechanism by which this occurs is unknown. We show that elongation factor 1 alpha (EF-1 alpha) is overexpressed in opaque2 endosperm compared with its normal counterpart and that there is a highly significant correlation between EF-1 alpha concentration and the total lysine content of the endosperm. This relationship is also true for two other cereals, sorghum and barley. It appears that genetic selection for genotypes with a high concentration of EF-1 alpha can significantly improve the nutritional quality of maize and other cereals. Images Fig. 1 Fig. 2 PMID:7567989

  11. Keggin-lysine hybrid nanostructures in the shape modulation of gold

    NASA Astrophysics Data System (ADS)

    Das, Subhasis; Ghosh, Tanmay; Satpati, Biswarup; Sanyal, Ambarish; Bala, Tanushree

    2014-03-01

    We show here that L-lysine effectively complexes with phosphomolybdic acid (PMA) and the solution mixture when added to a 10-3 M aqueous solution of HAuCl4 after UV-irradiation for 3 h leads to the slow reduction and consequent formation of gold nanotriangles with a high degree of anisotropy. The same reaction carried out in a 12.5 kDa cutoff dialysis bag where the irradiated PMA-lysine solution was kept inside and stirred in a beaker containing aqueous HAuCl4, did not lead to the formation of gold nanotriangles. This implies that L-lysine plays the role of a shape-modulating agent and hence this study proves an improvement in the understanding of the role of such organic-inorganic hybrid structures in the synthesis and growth of anisotropic nanoparticles.

  12. The lysine biosynthetic enzyme Lys4 influences iron metabolism, mitochondrial function and virulence in Cryptococcus neoformans.

    PubMed

    Do, Eunsoo; Park, Minji; Hu, Guanggan; Caza, Mélissa; Kronstad, James W; Jung, Won Hee

    2016-09-01

    The lysine biosynthesis pathway via α-aminoadipate in fungi is considered an attractive target for antifungal drugs due to its absence in mammalian hosts. The iron-sulfur cluster-containing enzyme homoaconitase converts homocitrate to homoisocitrate in the lysine biosynthetic pathway, and is encoded by LYS4 in the model yeast Saccharomyces cerevisiae. In this study, we identified the ortholog of LYS4 in the human fungal pathogen, Cryptococcus neoformans, and found that LYS4 expression is regulated by iron levels and by the iron-related transcription factors Hap3 and HapX. Deletion of the LYS4 gene resulted in lysine auxotrophy suggesting that Lys4 is essential for lysine biosynthesis. Our study also revealed that lysine uptake was mediated by two amino acid permeases, Aap2 and Aap3, and influenced by nitrogen catabolite repression (NCR). Furthermore, the lys4 mutant showed increased sensitivity to oxidative stress, agents that challenge cell wall/membrane integrity, and azole antifungal drugs. We showed that these phenotypes were due in part to impaired mitochondrial function as a result of LYS4 deletion, which we propose disrupts iron homeostasis in the organelle. The combination of defects are consistent with our observation that the lys4 mutant was attenuated virulence in a mouse inhalation model of cryptococcosis. PMID:27353379

  13. Critical lysine residues of Klf4 required for protein stabilization and degradation

    SciTech Connect

    Lim, Key-Hwan; Kim, So-Ra; Ramakrishna, Suresh; Baek, Kwang-Hyun

    2014-01-24

    Highlights: • Klf4 undergoes the 26S proteasomal degradation by ubiquitination on its multiple lysine residues. • Essential Klf4 ubiquitination sites are accumulated between 190–263 amino acids. • A mutation of lysine at 232 on Klf4 elongates protein turnover. • Klf4 mutants dramatically suppress p53 expression both under normal and UV irradiated conditions. - Abstract: The transcription factor, Krüppel-like factor 4 (Klf4) plays a crucial role in generating induced pluripotent stem cells (iPSCs). As the ubiquitination and degradation of the Klf4 protein have been suggested to play an important role in its function, the identification of specific lysine sites that are responsible for protein degradation is of prime interest to improve protein stability and function. However, the molecular mechanism regulating proteasomal degradation of the Klf4 is poorly understood. In this study, both the analysis of Klf4 ubiquitination sites using several Klf4 deletion fragments and bioinformatics predictions showed that the lysine sites which are signaling for Klf4 protein degradation lie in its N-terminal domain (aa 1–296). The results also showed that Lys32, 52, 232, and 252 of Klf4 are responsible for the proteolysis of the Klf4 protein. These results suggest that Klf4 undergoes proteasomal degradation and that these lysine residues are critical for Klf4 ubiquitination.

  14. A sulfonium cation intermediate in the mechanism of methionine sulfoxide reductase B: a DFT study.

    PubMed

    Robinet, Jesse J; Dokainish, Hisham M; Paterson, David J; Gauld, James W

    2011-07-28

    The hybrid density functional theory method B3LYP in combination with three systematically larger active site models has been used to investigate the substrate binding and catalytic mechanism by which Neisseria gonorrhoeae methionine sulfoxide reductase B (MsrB) reduces methionine-R-sulfoxide (Met-R-SO) to methionine. The first step in the overall mechanism is nucleophilic attack of an active site thiolate at the sulfur of Met-R-SO to form an enzyme-substrate sulfurane. This occurs with concomitant proton transfer from an active site histidine (His480) residue to the substrates oxygen center. The barrier for this step, calculated using our largest most complete active site model, is 17.2 kJ mol(-1). A subsequent conformational rearrangement and intramolecular -OH transfer to form an enzyme-derived sulfenic acid ((Cys495)S-OH) is not enzymatically feasible. Instead, transfer of a second proton from a second histidyl active site residue (His477) to the sulfurane's oxygen center to give water and a sulfonium cation intermediate is found to be greatly preferred, occurring with a quite low barrier of just 1.2 kJ mol(-1). Formation of the final product complex in which an intraprotein disulfide bond is formed with generation of methionine preferably occurs in one step via nucleophilic attack of the sulfur of a second enzyme thiolate ((Cys440)S(-)) at the S(Cys495) center of the sulfonium intermediate with a barrier of 23.8 kJ mol(-1). An alternate pathway for formation of the products via a sulfenic acid intermediate involves enzymatically feasible, but higher energy barriers. The role and impact of hydrogen bonding and active site residues on the properties and stability of substrate and mechanism intermediates and the affects of mutating His477 are also examined and discussed. PMID:21721538

  15. Evaluation of a diet dilution technique for measuring the response of broiler chickens to increasing concentrations of lysine.

    PubMed

    Gous, R M; Morris, T R

    1985-04-01

    Three experiments were conducted on male broiler chickens between one and three weeks of age to determine their response to dietary lysine concentrations. Serial dilutions of a summit diet shown to be first-limiting in lysine were fed in all experiments. The balance between amino acids in these diets was maintained within narrow limits. Intake of the most-limiting amino acid was the most important factor determining growth rate; protein intake as such was of little or no importance. The efficiency of utilisation of dietary lysine for protein growth was calculated to be 65.05 mg/g protein gain, representing a net efficiency of 0.85. The diet dilution technique overcomes the major disadvantage of the graded supplementation method for determining the requirements of amino acids, namely that of the amino acid balance changing systematically in successive dietary treatments.

  16. Effect of irradiation on Nε-carboxymethyl-lysine and Nε-carboxyethyl-lysine formation in cooked meat products during storage

    NASA Astrophysics Data System (ADS)

    Yu, Ligang; He, Zhiyong; Zeng, Maomao; Zheng, Zongping; Chen, Jie

    2016-03-01

    This study investigated the effects of irradiation on Nε-carboxymethyl-lysine (CML) and Nε-carboxyethyl-lysine (CEL) formation in cooked red and white meats during storage. The results showed that irradiation did not affect CML/CEL formation (0 weeks). After 6 weeks, CML/CEL contents in the irradiated samples exhibited a higher growth rate than the non-irradiated samples, especially the red meat. The results of electron spin resonance spectrometry and 2-Thiobarbituric acid-reactive substances suggested irradiation had induced free-radical reactions and accelerated lipid oxidation during storage. A linear correlation (r=0.810-0.906, p<0.01) was found between the loss of polyunsaturated fatty acids content and increase of CML/CEL content in the irradiated samples after 0 and 6 weeks of storage. The results indicate that irradiation-induced lipid oxidation promotes CML/CEL formation, and CML/CEL formation by the lipid oxidation pathways may be an important pathway for CML/CEL accumulation in irradiated meat products during storage.

  17. N-formylation of lysine in histone proteins as a secondary modification arising from oxidative DNA damage

    PubMed Central

    Jiang, Tao; Zhou, Xinfeng; Taghizadeh, Koli; Dong, Min; Dedon, Peter C.

    2007-01-01

    The posttranslational modification of histone and other chromatin proteins has a well recognized but poorly defined role in the physiology of gene expression. With implications for interfering with these epigenetic mechanisms, we now report the existence of a relatively abundant secondary modification of chromatin proteins, the N6-formylation of lysine that appears to be uniquely associated with histone and other nuclear proteins. Using both radiolabeling and sensitive bioanalytical methods, we demonstrate that the formyl moiety of 3′-formylphosphate residues arising from 5′-oxidation of deoxyribose in DNA, caused by the enediyne neocarzinostatin, for example, acylate the N6-amino groups of lysine side chains. A liquid chromatography (LC)–tandem mass spectrometry (MS) method was developed to quantify the resulting N6-formyl-lysine residues, which were observed to be present in unperturbed cells and all sources of histone proteins to the extent of 0.04–0.1% of all lysines in acid-soluble chromatin proteins including histones. Cells treated with neocarzinostatin showed a clear dose–response relationship for the formation of N6-formyl-lysine, with this nucleosome linker-selective DNA-cleaving agent causing selective N6-formylation of the linker histone H1. The N6-formyl-lysine residue appears to represent an endogenous histone secondary modification, one that bears chemical similarity to lysine N6-acetylation recognized as an important determinant of gene expression in mammalian cells. The N6-formyl modification of lysine may interfere with the signaling functions of lysine acetylation and methylation and thus contribute to the pathophysiology of oxidative and nitrosative stress. PMID:17190813

  18. Age-related changes of muscle and plasma amino acids in healthy children.

    PubMed

    Hammarqvist, Folke; Angsten, Gertrud; Meurling, Staffan; Andersson, Kerstin; Wernerman, Jan

    2010-07-01

    The aim of the study was to explore if changes in muscle and plasma amino acid concentrations developed during growth and differed from levels seen in adults. The gradient and concentrations of free amino acids in muscle and plasma were investigated in relation to age in metabolic healthy children. Plasma and specimens from the abdominal muscle were obtained during elective surgery. The children were grouped into three groups (group 1: < 1 year, n = 8; group 2: 1-4 years, n = 13 and group 3: 5-15 years, n = 15). A reference group of healthy adults (21-38 years, n = 22) was included in their comparisons and reflected specific differences between children and adults. In muscle the concentrations of 8 out of 19 amino acids analysed increased with age, namely taurine, aspartate, threonine, alanine, valine, isoleucine, leucine, histidine, as well as the total sums of branched chain amino acids (BCAA), basic amino acids (BAA) and total sum of amino acids (P < 0.05). In plasma the concentrations of threonine, glutamine, valine, cysteine, methionine, leucine, lysine, tryptophane, arginine, BCAA, BAA and the essential amino acids correlated with age (P < 0.05). These results indicate that there is an age dependency of the amino acid pattern in skeletal muscle and plasma during growth.

  19. Proton Affinity of Isomeric Dipeptides Containing Lysine and Non-Proteinogenic Lysine Homologues.

    PubMed

    Batoon, Patrick; Ren, Jianhua

    2016-08-18

    Conformational effects on the proton affinity of oligopeptides have been studied using six alanine (A)-based acetylated dipeptides containing a basic probe that is placed closest to either the C- or the N-terminus. The basic probe includes Lysine (Lys) and two nonproteinogenic Lys-homologues, ornithine (Orn) and 2,3-diaminopropionic acid (Dap). The proton affinities of the peptides have been determined using the extended Cooks kinetic method in a triple quadrupole mass spectrometer. Computational studies have been carried out to search for the lowest energy conformers and to calculate theoretical proton affinities as well as various molecular properties using the density functional theory. The dipeptides containing a C-terminal probe, ALys, AOrn, and ADap, were determined to have a higher proton affinity by 1-4 kcal/mol than the corresponding dipeptides containing an N-terminal probe, LysA, OrnA, and DapA. For either the C-probe peptides or the N-probe peptides, the proton affinity reduces systematically as the side-chain of the probe residue is shortened. The difference in the proton affinities between isomeric peptides is largely associated with the variation of the conformations. The peptides with higher values of the proton affinity adopt a relatively compact conformation such that the protonated peptides can be stabilized through more efficient internal solvation. PMID:27459294

  20. Class I Lysine Deacetylases Facilitate Glucocorticoid-induced Transcription*

    PubMed Central

    Kadiyala, Vineela; Patrick, Nina M.; Mathieu, Wana; Jaime-Frias, Rosa; Pookhao, Naruekamol; An, Lingling; Smith, Catharine L.

    2013-01-01

    Nuclear receptors use lysine acetyltransferases and lysine deacetylases (KDACs) in regulating transcription through histone acetylation. Lysine acetyltransferases interact with steroid receptors upon binding of an agonist and are recruited to target genes. KDACs have been shown to interact with steroid receptors upon binding to an antagonist. We have shown previously that KDAC inhibitors (KDACis) potently repress the mouse mammary tumor virus promoter through transcriptional mechanisms and impair the ability of the glucocorticoid receptor (GR) to activate it, suggesting that KDACs can play a positive role in GR transactivation. In the current study, we extended this analysis to the entire GR transcriptome and found that the KDACi valproic acid impairs the ability of agonist-bound GR to activate about 50% of its target genes. This inhibition is largely due to impaired transcription rather than defective GR processing and was also observed using a structurally distinct KDACi. Depletion of KDAC1 expression mimicked the effects of KDACi in over half of the genes found to be impaired in GR transactivation. Simultaneous depletion of KDACs 1 and 2 caused full or partial impairment of several more GR target genes. Altogether we found that Class I KDAC activity facilitates GR-mediated activation at a sizable fraction of GR-activated target genes and that KDAC1 alone or in coordination with KDAC2 is required for efficient GR transactivation at many of these target genes. Finally, our work demonstrates that KDACi e